U.S. patent application number 13/669130 was filed with the patent office on 2013-08-22 for dig-3 insecticidal cry toxins.
This patent application is currently assigned to Dow Agrosciences LLC. The applicant listed for this patent is Dow Agrosciences LLC. Invention is credited to Holly Jean Butler, Justin M. LIRA, Thomas Meade, Kenneth Narva, Doug A. Smith.
Application Number | 20130219570 13/669130 |
Document ID | / |
Family ID | 42269529 |
Filed Date | 2013-08-22 |
United States Patent
Application |
20130219570 |
Kind Code |
A1 |
LIRA; Justin M. ; et
al. |
August 22, 2013 |
DIG-3 INSECTICIDAL Cry TOXINS
Abstract
DIG-3 Cry toxins, polynucleotides encoding such toxins, and
transgenic plants that produce such toxins are useful to control
insect pests.
Inventors: |
LIRA; Justin M.;
(Zionsville, IN) ; Butler; Holly Jean;
(Indianapolis, IN) ; Smith; Doug A.; (Noblesville,
IN) ; Narva; Kenneth; (Zionsville, IN) ;
Meade; Thomas; (Zionsville, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow Agrosciences LLC; |
|
|
US |
|
|
Assignee: |
Dow Agrosciences LLC
Zionsville
IN
|
Family ID: |
42269529 |
Appl. No.: |
13/669130 |
Filed: |
November 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12730295 |
Mar 24, 2010 |
8304604 |
|
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13669130 |
|
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61170189 |
Apr 17, 2009 |
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Current U.S.
Class: |
800/302 ;
435/412; 435/419; 514/4.5 |
Current CPC
Class: |
Y02A 40/162 20180101;
C07K 14/325 20130101; Y02A 40/146 20180101; C12N 15/8286 20130101;
A01N 43/50 20130101 |
Class at
Publication: |
800/302 ;
514/4.5; 435/419; 435/412 |
International
Class: |
A01N 43/50 20060101
A01N043/50; C12N 15/82 20060101 C12N015/82 |
Claims
1. A transgenic plant cell comprising a nucleic acid molecule that
encodes an isolated polypeptide comprising a core toxin segment
having at least 97% sequence identity to the amino acid sequence of
residues 73 to 643 of SEQ ID NO:2, wherein said polypeptide has
insecticidal activity, and wherein said plant cell also produces an
insecticidal protein selected from the group consisting of a Cry1F
protein and a Cry1A protein.
2. The plant cell of claim 1, wherein said cell is selected from
the group consisting of a corn plant cell and a vegetable plant
cell.
3. A method of controlling a lepidopteran, said method comprising
providing an isolated polypeptide to said lepidopteran, said
polypeptide comprising a core toxin segment having at least 97%
sequence identity to the amino acid sequence of residues 73 to 643
of SEQ ID NO:2, wherein said polypeptide has insecticidal activity,
wherein said method further comprises providing an insecticidal
protein to said lepidopteran, said protein being selected from the
group consisting of a Cry1F protein and a Cry1A protein, and
wherein said lepidopteran is selected from the group consisting of
a European corn borer that is resistant to Cry1F, and a diamond
back moth that is resistant to a Cry1A.
4. The method of claim 3, wherein said lepidopteran is said
European corn borer, and said insecticidal protein is said Cry1F
protein.
5. The method of claim 3, wherein said lepidopteran is said diamond
back moth, and said insecticidal protein is said Cry1A protein.
6. The method of claim 3, where said method is used to control a
pest population, wherein said pest population is selected from the
group consisting of a population of European corn borer that is
resistant to Cry1F, and a diamond back moth that is resistant to a
Cry1A.
7. The method of claim 6, wherein said population is said
population of European corn borer, and said insecticidal protein is
said Cry1F protein.
8. The method of claim 6, wherein said population is said
population of diamond back moth, and said insecticidal protein is
said Cry1A protein.
9. A transgenic plant comprising a plurality of cells of claim
1.
10. The plant cell of claim 1, wherein said insecticidal protein is
said Cry1F protein.
11. The plant cell of claim 1, wherein said insecticidal protein is
said Cry1A protein.
12. The plant cell of claim 11, wherein said Cry1A protein is
selected from the group consisting of a Cry1Ab protein and a Cry1Ac
protein.
13. The method of claim 5, wherein said Cry1A protein is selected
from the group consisting of a Cry1Ab protein and a Cry1Ac
protein.
14. The method of claim 8, wherein said Cry1A protein selected from
the group consisting of a Cry1Ab protein and a Cry1Ac protein.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/730,295, filed Mar. 24, 2010, which claims
benefit of U.S. Provisional Patent Application No. 61/170,189,
filed Apr. 17, 2009, the disclosures of which are expressly
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] This invention concerns new insecticidal Cry toxins and
their use to control insects.
BACKGROUND OF THE INVENTION
[0003] Bacillus thuringiensis (B.t.) is a soil-borne bacterium that
produces pesticidal crystal proteins known as delta endotoxins or
Cry proteins. Cry proteins are oral intoxicants that function by
acting on midgut cells of susceptible insects. An extensive list of
delta endotoxins is maintained and regularly updated at
http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html.
[0004] European corn borer (ECB), Ostrinia nubilalis (Hubner), is
the most damaging insect pest of corn throughout the United States
and Canada, and causes an estimated $1 billion revenue loss each
year due to crop yield loss and expenditures for insect management
(Witkowski et al., 2002). Transgenic corn expressing genes encoding
Cry proteins, most notably Cry1Ab, Cry1Ac, or Cry1F, provide
commercial levels of efficacy against ECB.
[0005] Despite the success of ECB-resistant transgenic corn, the
possibility of the development of resistant insect populations
threatens the long-term durability of Cry proteins in ECB control
and creates the need to discover and develop new Cry proteins to
control ECB and other pests. Insect resistance to B.t. Cry proteins
can develop through several mechanisms (Heckel et al., 2007, Pigott
and Ellar, 2007). Multiple receptor protein classes for Cry
proteins have been identified within insects, and multiple examples
exist within each receptor class. Resistance to a particular Cry
protein may develop, for example, by means of a mutation within the
toxin-binding portion of a cadherin domain of a receptor protein. A
further means of resistance may be mediated through a
protoxin-processing protease. Thus, resistance to Cry toxins in
species of Lepidoptera has a complex genetic basis, with at least
four distinct, major resistance genes. Lepidopteran insects
resistant to Cry proteins have developed in the field within the
species Plutella xylostella (Tabashnik, 1994), Trichoplusia ni
(Janmaat and Myers 2003, 2005), and Helicoverpa zeae (Tabashnik et
al., 2008). Development of new high potency Cry proteins would
provide additional tools for management of ECB and other insect
pests. Cry proteins with different modes of action produced in
combination in transgenic corn would prevent the development ECB
insect resistance and protect the long term utility of B.t.
technology for insect pest control.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides insecticidal Cry toxins,
including the toxin designated herein as DIG-3 as well as variants
of DIG-3, nucleic acids encoding these toxins, methods of
controlling pests using the toxins, methods of producing the toxins
in transgenic host cells, and transgenic plants that produce the
toxins. The predicted amino acid sequence of the wild type DIG-3
toxin is given in SEQ ID NO:2.
[0007] As described in Example 1, a nucleic acid encoding the DIG-3
protein was isolated from a B.t. strain internally designated by
Dow AgroSciences LLC as PS46L. The nucleic acid sequence for the
full length coding region was determined, and the full length
protein sequence was deduced from the nucleic acid sequence. The
DIG-3 toxin has some similarity to Cry1BII (Genbank Accession No.
AAM93496) and other B. thuringiensis Cry1B-type proteins
(http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html).
[0008] Insecticidally active variants of the DIG-3 toxin are also
described herein, and are referred to collectively as DIG-3
toxins.
[0009] DIG-3 toxins may also be used in combination with RNAi
methodologies for control of other insect pests. For example, DIG-3
can be used in transgenic plants in combination with a dsRNA for
suppression of an essential gene in corn rootworm or an essential
gene in an insect pest. Such target genes include, for example,
vacuolar ATPase, ARF-1, Act42A, CHD3, EF-1a, and TFIIB. An example
of a suitable target gene is vacuolar ATPase, as disclosed in
WO2007/035650.
[0010] A surprising finding reported herein is that DIG-3 toxins
are active against populations of European corn borer and diamond
back moth that are resistant to Cry1F and Cry1A toxins.
Accordingly, DIG-3 toxins are ideal candidates for use to control
of Lepidopteran pests. The toxins can be used alone or in
combination with other Cry toxins, such as Cry1F, Cry1Ab, and
Cry1Ac, to control development of resistant insect populations.
[0011] Insecticidally active fragments of SEQ ID NO:2, and
nucleotides encoding such fragments, are another aspect of the
invention.
[0012] In one embodiment the invention provides an isolated DIG-3
toxin polypeptide comprising a core toxin segment selected from the
group consisting of [0013] (a) a polypeptide comprising the amino
acid sequence of residues 113 to 643 of SEQ ID NO:2;
[0014] (b) a polypeptide comprising an amino acid sequence having
at least 90% sequence identity to the amino acid sequence of
residues 113 to 643 of SEQ ID NO:2;
[0015] (c) a polypeptide comprising an amino acid sequence of
residues 113 to 643 of SEQ ID NO:2 with up to 20 amino acid
substitutions, deletions, or modifications that do not adversely
affect expression or activity of the toxin encoded by SEQ ID
NO:2.
[0016] In one embodiment the invention provides an isolated DIG-3
toxin polypeptide comprising a core toxin segment selected from the
group consisting of [0017] (a) a polypeptide comprising the amino
acid sequence of residues 73 to 643 of SEQ ID NO:2;
[0018] (b) a polypeptide comprising an amino acid sequence having
at least 90% sequence identity to the amino acid sequence of
residues 73 to 643 of SEQ ID NO:2;
[0019] (c) a polypeptide comprising an amino acid sequence of
residues 73 to 643 of SEQ ID NO:2 with up to 20 amino acid
substitutions, deletions, or modifications that do not adversely
affect expression or activity of the toxin encoded by SEQ ID
NO:2.
[0020] In another embodiment the invention provides an isolated
DIG-3 toxin polypeptide comprising a DIG-3 core toxin segment
selected from the group consisting of [0021] (a) a polypeptide
comprising the amino acid sequence of residues 1 to 643 of SEQ ID
NO:2;
[0022] (b) a polypeptide comprising an amino acid sequence having
at least 90% sequence identity to the amino acid sequence of
residues 1 to 643 of SEQ ID NO:2; [0023] (c) a polypeptide
comprising an amino acid sequence of residues 1 to 643 of SEQ ID
NO:2 with up to 20 amino acid substitutions, deletions, or
modifications that do not adversely affect expression or activity
of the toxin encoded by SEQ ID NO:2.
[0024] By "isolated" applicants mean that the polypeptide or DNA
molecules have been removed from their native environment and have
been placed in a different environment by the hand of man.
[0025] In another embodiment the invention provides a plant
comprising a DIG-3 toxin.
[0026] In another embodiment the invention provides a method for
controlling a pest population comprising contacting said population
with a pesticidally effective amount of a DIG-3 toxin.
[0027] In another embodiment the invention provides an isolated
nucleic acid that encodes a DIG-3 toxin.
[0028] In another embodiment the invention provides a DNA construct
comprising a nucleotide sequence that encodes a DIG-3 toxin
operably linked to a promoter that is not derived from Bacillus
thuringiensis and is capable of driving expression in a plant. The
invention also provides a transgenic plant that comprises the DNA
construct stably incorporated into its genome and a method for
protecting a plant from a pest comprising introducing the construct
into said plant.
BRIEF DESCRIPTION OF THE SEQUENCES
[0029] SEQ ID NO:1 DNA sequence encoding full-length DIG-3 toxin;
3771 nt.
[0030] SEQ ID NO:2 Full-length DIG-3 protein sequence; 1256 aa.
[0031] SEQ ID NO:3 Plant-optimized full length DIG-3 DNA sequence;
3771 nt.
[0032] SEQ ID NO:4 Cry1Ab protoxin segment; 545 aa.
[0033] SEQ ID NO:5 Chimeric toxin: DIG-3 Core toxin segment/Cry1Ab
protoxin segment; 1188 aa.
[0034] SEQ ID NO:6 Dicot-optimized DNA sequence encoding the Cry1Ab
protoxin segment; 1635 nt SEQ ID NO:7 Maize-optimized DNA sequence
encoding the Cry1Ab protoxin segment; 1635 nt
DETAILED DESCRIPTION OF THE INVENTION
[0035] DIG-3 Toxins, and Insecticidally Active Variants.
[0036] In addition to the full length DIG-3 toxin of SEQ ID NO:2,
the invention encompasses insecticidally active variants. By the
term "variant", applicants intend to include fragments, certain
deletion and insertion mutants, and certain fusion proteins. DIG-3
is a classic three-domain Cry toxin. As a preface to describing
variants of the DIG-3 toxin that are included in the invention, it
will be useful to briefly review the architecture of three-domain
Cry toxins in general and of the DIG-3 protein toxin in
particular.
[0037] A majority of Bacillus thuringiensis delta-endotoxin crystal
protein molecules are composed of two functional segments. The
protease-resistant core toxin is the first segment and corresponds
to about the first half of the protein molecule. The full
.about.130 kDa protoxin molecule is rapidly processed to the
resistant core segment by proteases in the insect gut. The segment
that is deleted by this processing will be referred to herein as
the "protoxin segment". The protoxin segment is believed to
participate in toxin crystal formation (Arvidson et al., 1989). The
protoxin segment may thus convey a partial insect specificity for
the toxin by limiting the accessibility of the core to the insect
by reducing the protease processing of the toxin molecule (Haider
et al., 1986) or by reducing toxin solubility (Aronson et al.,
1991). B.t. toxins, even within a certain class, vary to some
extent in length and in the precise location of the transition from
the core toxin segment to protoxin segment. The transition from
core toxin segment to protoxin segment will typically occur at
between about 50% to about 60% of the full length toxin. SEQ ID
NO:2 discloses the 1256 amino acid sequence of the full-length
DIG-3 polypeptide, of which the N-terminal 643 amino acids comprise
the DIG-3 core toxin segment. The 5'-terminal 1929 nucleotides of
SEQ ID NO:1 comprise the coding region for the core toxin
segment.
[0038] Three dimensional crystal structures have been determined
for Cry1 Aa1, Cry2Aa1, Cry3Aa1, Cry3Bb1, Cry4Aa, Cry4Ba and
Cry8Ea1. These structures for the core toxins are remarkably
similar and are comprised of three distinct domains with the
features described below (reviewed in de Maagd et al., 2003).
[0039] Domain I is a bundle of seven alpha helices where
.alpha.-helix 5 is surrounded by six amphipathic helices. This
domain has been implicated in pore formation and shares homology
with other pore forming proteins including hemolysins and colicins.
Domain I of the DIG-3 protein comprises amino acid residues 56 to
278 of SEQ ID NO:2.
[0040] Domain II is formed by three anti-parallel beta sheets
packed together in a beta prism. The loops of this domain play
important roles in binding insect midgut receptors. In Cry1A
proteins, surface exposed loops at the apices of Domain II beta
sheets are involved in binding to Lepidopteran cadherin receptors.
Cry3Aa Domain II loops bind a membrane-associated metalloprotease
of Leptinotarsa decemlineata (Say) (Colorado potato beetle) in a
similar fashion (Ochoa-Campuzano et al., 2007). Domain II shares
homology with certain carbohydrate-binding proteins including
vitelline and jacaline. Domain II of the DIG-3 protein comprises
amino acid residues 283 to 493 of SEQ ID NO:2.
[0041] Domain 111 is a beta sandwich of two anti-parallel beta
sheets. Structurally this domain is related to carbohydrate-binding
domains of proteins such as glucanases, galactose oxidase,
sialidase and others. Domain III binds certain classes of receptor
proteins and perhaps participates in insertion of an oligomeric
toxin pre-pore that interacts with a second class of receptors,
examples of which are aminopeptidase and alkaline phosphatase in
the case of Cry1A proteins (Pigott and Ellar, 2007). Analogous Cry
Domain III receptors have yet to be identified in Coleoptera.
Conserved B.t. sequence blocks 2 and 3 map near the N-terminus and
C-terminus of Domain 2, respectively. Hence, these conserved
sequence blocks 2 and 3 are approximate boundary regions between
the three functional domains. These regions of conserved DNA and
protein homology have been exploited for engineering recombinant
B.t. toxins (U.S. Pat. No. 6,090,931, WO 91/01087, WO 95/06730, WO
1998022595). Domain III of the DIG-3 protein comprises amino acid
residues 503 to 641 of SEQ ID NO:2.
[0042] It has been reported that .alpha.-helix 1 of Domain I is
removed following receptor binding. Aronson et al. (1999)
demonstrated that Cry1Ac bound to BBMV was protected from
proteinase K cleavage beginning at residue 59, just after
.alpha.-helix 1; similar results were cited for Cry1 Ab. Gomez et
al. (2002) found that Cry1Ab oligomers formed upon BBMV receptor
binding lacked the .alpha.-helix 1 portion of Domain I. Also,
Soberon et al. (2007) have shown that N-terminal deletion mutants
of Cry1 Ab and Cry1Ac which lack approximately 60 amino acids
encompassing .alpha.-helix 1 on the three dimensional Cry structure
are capable of assembling monomers of molecular weight about 60 kDa
into pre-pores in the absence of cadherin binding. These N-terminal
deletion mutants were reported to be active on Cry-resistant insect
larvae. Furthermore, Diaz-Mendoza et al. (2007) described Cry1Ab
fragments of 43 kDa and 46 kDa that retained activity on
Mediterranean corn borer (Sesamia nonagrioides). These fragments
were demonstrated to include amino acid residues 116 to 423;
however the precise amino acid sequences were not elucidated and
the mechanism of activity of these proteolytic fragments is
unknown. The results of Gomez et al. (2002), Soberon et al. (2007),
and Diaz-Mendoza et al. (2007) contrast with those of Hofte et al.
(1986), who reported that deletion of 36 amino acids from the
N-terminus of Cry1Ab resulted in loss of insecticidal activity.
[0043] We have deduced the beginnings and ends of .alpha.-helix 1,
.alpha.-helix 2A, .alpha.-helix 2B, and .alpha.-helix 3, and the
location of the spacer regions between them in Domain I of the
DIG-3 toxin by comparing the DIG-3 protein sequence with the
protein sequence for Cry8Ea1, for which the structure is known.
These locations are described in Table 1.
TABLE-US-00001 TABLE 1 Amino acid coordinates of projected
.alpha.-helices of DIG-3 protein. .alpha.-helix 1 spacer
.alpha.-helix 2A spacer .alpha.-helix 2B spacer .alpha.-helix 3
Residues of 53-70 71-76 77-91 92-99 100-108 109-113 114-138 SEQ ID
NO: 2
[0044] Amino terminal deletion variants of DIG-3. In one of its
aspects the invention provides DIG-3 variants in which all or part
of .alpha.-helix 1, .alpha.-helix 2A, and .alpha.-helix 2B are
deleted to improve insecticidal activity and avoid development of
resistance by insects. These modifications are made to provide
DIG-3 variants with improved attributes, such as improved target
pest spectrum, potency, and insect resistance management. In some
embodiments of the invention, the subject modifications may affect
the efficiency of protoxin activation and pore formation, leading
to insect intoxication. More specifically, to provide DIG-3
variants with improved attributes, step-wise deletions are
described that remove part of the nucleic acid sequence encoding
the N-terminus of the DIG-3 protein. The deletions remove all of
.alpha.-helix 1 and all or part of .alpha.-helix 2 in Domain I,
while maintaining the structural integrity of a-helices 3 through
7. The subject invention therefore relates in part to improvements
to Cry protein efficacy made by engineering the a-helical
components of Domain 1 for more efficient pore formation. More
specifically, the subject invention relates in part to improved
DIG-3 proteins designed to have N-terminal deletions in regions
with putative secondary structure homology to .alpha.-helix 1 and
.alpha.-helix 2 in Domain I of Cry1 proteins.
[0045] Deletions to improve the insecticidal properties of the
DIG-3 toxins may initiate before the predicted .alpha.-helix 2A
start, and may terminate after the .alpha.-helix 2B end, but
preferably do not extend into .alpha.-helix 3.
[0046] In designing coding sequences for the N-terminal deletion
variants, an ATG start codon, encoding methionine, is inserted at
the 5' end of the nucleotide sequence designed to encode the
deletion variant. For sequences designed for use in transgenic
plants, it may be of benefit to adhere to the "N-end rule" of
Varshaysky (1997). It is taught that some amino acids may
contribute to protein instability and degradation in eukaryotic
cells when displayed as the N-terminal residue of a protein. For
example, data collected from observations in yeast and mammalian
cells indicate that the N-terminal destabilizing amino acids are F,
L, W, Y, R, K, H, I, N, Q, D, E and possibly P. While the specifics
of protein degradation mechanisms may differ somewhat between
organisms, the conservation of identity of N-terminal destabilizing
amino acids seen above suggests that similar mechanisms may
function in plant cells. For instance, Worley et al. (1998) found
that in plants, the N-end rule includes basic and aromatic
residues. It is a possibility that proteolytic cleavage by plant
proteases near the start of .alpha.-helix 3 of subject B.t.
insecticidal proteins may expose a destabilizing N-terminal amino
acid. Such processing may target the cleaved proteins for rapid
decay and limit the accumulation of the B.t. insecticidal proteins
to levels insufficient for effective insect control. Accordingly,
for N-terminal deletion variants that begin with one of the
destabilizing amino acids, applicants prefer to add a codon that
specifies a G (glycine) amino acid between the translational
initiation methionine and the destabilizing amino acid.
[0047] Example 2 gives specific examples of amino-terminal deletion
variants of DIG-3 in accordance with the invention. Additional
useful fragments that retain toxicity can be identified by trypsin
or chymotrypsin digestion of the full length solubilized crystal
protein. Further examples of toxic DIG-3 protein fragments may be
encoded by fragments of the DIG-3 coding region. Insect active
DIG-3 variants will mostly have a short N-terminal truncation and a
long C-terminal truncation. The N-terminal end of the smallest
toxic fragment is conveniently determined by N-terminal amino acid
sequence determination of trypsin- or chymotrypsin-treated soluble
crystal protein by techniques routinely available in the art.
[0048] Chimeric Toxins.
[0049] Chimeric proteins utilizing the core toxin segment of one
Cry toxin fused to the protoxin segment of another Cry toxin have
previously been reported. DIG-3 variants include toxins comprising
an N-terminal core toxin segment of a DIG-3 toxin (which may be
full length or have the N-terminal deletions described above) fused
to a heterologous protoxin segment at some point past the end of
the core toxin segment. The transition to the heterologous protoxin
segment can occur at approximately the native core toxin/protoxin
junction or, in the alternative, a portion of the native protoxin
(extending past the core toxin segment) can be retained with the
transition to the heterologous protoxin occurring downstream. As an
example, a chimeric toxin of the subject invention has the full
core toxin segment of DIG-3 (amino acids 1-643) and a heterologous
protoxin segment (amino acids 643 to the C-terminus). In a
preferred embodiment, the heterologous protoxin segment is derived
from a Cry1Ab delta-endotoxin, as illustrated in SEQ ID NO:5.
[0050] SEQ ID NO:4 discloses the 545 amino acid sequence of a
Cry1Ab protoxin segment useful in DIG-3 variants of the invention.
Attention is drawn to the last about 100 to 150 amino acids of this
protoxin segment, which it is most critical to include in the
chimeric toxin of the subject invention.
[0051] Protease Sensitivity Variants.
[0052] Insect gut proteases typically function in aiding the insect
in obtaining needed amino acids from dietary protein. The best
understood insect digestive proteases are serine proteases, which
appear to be the most common type (Englemann and Geraerts (1980),
particularly in Lepidopteran species. Coleopteran insects have guts
that are more neutral to acidic than are Lepidopteran guts. The
majority of Coleopteran larvae and adults, for example Colorado
potato beetle, have slightly acidic midguts, and cysteine proteases
provide the major proteolytic activity (Wolfson and Murdock, 1990).
More precisely, Thie and Houseman (1990) identified and
characterized the cysteine proteases, cathepsin B-like and
cathepsin H-like, and the aspartyl protease, cathepsin D-like, in
Colorado potato beetle. Gillikin et al. (1992) characterized the
proteolytic activity in the guts of western corn rootworm larvae
and found primarily cysteine proteases. U.S. Pat. No. 7,230,167
disclosed that a protease activity attributed to cathepsin G exists
in western corn rootworm. The diversity and different activity
levels of the insect gut proteases may influence an insect's
sensitivity to a particular B.t. toxin.
[0053] In another embodiment of the invention, protease cleavage
sites may be engineered at desired locations to affect protein
processing within the midgut of susceptible larvae of certain
insect pests. These protease cleavage sites may be introduced by
methods such as chemical gene synthesis or splice overlap PCR
(Horton et al., 1989). Serine protease recognition sequences, for
example, can optionally be inserted at specific sites in the Cry
protein structure to effect protein processing at desired deletion
points within the midgut of susceptible larvae. Serine proteases
that can be exploited in such fashion include Lepidopteran midgut
serine proteases such as trypsin or trypsin-like enzymes,
chymotrypsin, elastase, etc. (Christeller et al., 1992). Further,
deletion sites identified empirically by sequencing Cry protein
digestion products generated with unfractionated larval midgut
protease preparations or by binding to brush border membrane
vesicles can be engineered to effect protein activation. Modified
Cry proteins generated either by gene deletion or by introduction
of protease cleavage sites have improved activity on Lepidopteran
pests including Ostrinia nubilalis, Diatraea grandiosella,
Helicoverpa zea, Agrotis ipsilon, Spodoptera frugiperda, Spodoptera
exigua, Diatraea saccharalis, Loxagrotis albicosta, and other
target pests.
[0054] Coleopteran serine proteases such as trypsin, chymotrypsin
and cathepsin G-like protease, Coleopteran cysteine proteases such
as cathepsins (B-like, L-like, O-like, and K-like proteases) (Koiwa
et al., (2000) and Bown et al., (2004)], Coleopteran
metalloproteases such as ADAM10 [Ochoa-Campuzano et al., (2007)),
and Coleopteran aspartic acid proteases such as cathepsins D-like
and E-like, pepsin, plasmepsin, and chymosin may further be
exploited by engineering appropriate recognition sequences at
desired processing sites to affect Cry protein processing within
the midgut of susceptible larvae of certain insect pests.
[0055] A preferred location for the introduction of such protease
cleavage sites may be within the "spacer" region between
.alpha.-helix 2B and .alpha.-helix 3, for example within amino
acids 109 to 113 of the full length DIG-3 protein (SEQ ID NO:2 and
Table 1). Modified Cry proteins generated either by gene deletion
or by introduction of protease cleavage sites have improved
activity on insect pests including but not limited to western corn
rootworm, southern corn root worn, northern corn rootworm, and the
like.
[0056] Various technologies exist to enable determination of the
sequence of the amino acids which comprise the N-terminal or
C-terminal residues of polypeptides. For example, automated Edman
degradation methodology can be used in sequential fashion to
determine the N-terminal amino acid sequence of up to 30 amino acid
residues with 98% accuracy per residue. Further, determination of
the sequence of the amino acids comprising the carboxy end of
polypeptides is also possible [Bailey et al., (1992); U.S. Pat. No.
6,046,053]. Thus, in some embodiments, B.t. Cry proteins which have
been activated by means of proteolytic processing, for example, by
proteases prepared from the gut of an insect, may be characterized
and the N-terminal or C-terminal amino acids of the activated toxin
fragment identified. DIG-3 variants produced by introduction or
elimination of protease processing sites at appropriate positions
in the coding sequence to allow, or eliminate, proteolytic cleavage
of a larger variant protein by insect, plant or microorganism
proteases are within the scope of the invention. The end result of
such manipulation is understood to be the generation of toxin
fragment molecules having the same or better activity as the intact
(full length) toxin protein.
[0057] Domains of the DIG-3 Toxin.
[0058] The separate domains of the DIG-3 toxin, (and variants that
are 90%, 95%, or 97% identical to such domains) are expected to be
useful in forming combinations with domains from other Cry toxins
to provide new toxins with increased spectrum of pest toxicity,
improved potency, or increased protein stability. Domain I of the
DIG-3 protein consists of amino acid residues 56 to 278 of SEQ ID
NO:2. Domain II of the DIG-3 protein consists of amino acid
residues 283 to 493 of SEQ ID NO:2. Domain III of the DIG-3 protein
consists of amino acid residues 503 to 641 of SEQ ID NO:2. Domain
swapping or shuffling is a mechanism for generating altered
delta-endotoxin proteins. Domains II and III may be swapped between
delta-endotoxin proteins, resulting in hybrid or chimeric toxins
with improved pesticidal activity or target spectrum. Domain II is
involved in receptor binding, and the DIG-3 Domain II is very
divergent from other Cry1B toxins. Domain III binds certain classes
of receptor proteins and perhaps participates in insertion of an
oligomeric toxin pre-pore. Some Domain III substitutions in other
toxins have been shown to produce superior toxicity against
Spodoptera exigua (de Maagd et al., 1996), and guidance exists on
the design of the Cry toxin domain swaps (Knight et al., 2004).
[0059] Methods for generating recombinant proteins and testing them
for pesticidal activity are well known in the art (see, for
example, Naimov et al., (2001), de Maagd et al., (1996), Ge et al.,
(1991), Schnepf et al., (1990), Rang et al., (1999)). Domain I from
Cry1A and Cry3A proteins has been studied for the ability to insert
and form pores in membranes. .alpha.-helix 4 and .alpha.-helix 5 of
Domain I play key roles in membrane insertion and pore formation
[Walters et al., (1993), Gazit et al., (1998); Nunez-Valdez et al.,
(2001)], with the other alpha helices proposed to contact the
membrane surface like the ribs of an umbrella (Bravo et al.,
(2007); Gazit et al., (1998)).
[0060] DIG-3 Variants Created by Making a Limited Number of Amino
Acid Deletions, Substitutions, or Additions.
[0061] Amino acid deletions, substitutions, and additions to the
amino acid sequence of SEQ ID NO:2 can readily be made in a
sequential manner and the effects of such variations on
insecticidal activity can be tested by bioassay. Provided the
number of changes is limited in number, such testing does not
involve unreasonable experimentation. The invention includes
insecticidally active variants of the core toxin segment (amino
acids 1-643 of SEQ ID NO:2, or amino acids 73-643 of SEQ ID NO:2)
in which up to 10, up to 15, or up to 20 independent amino acid
additions, deletions, or substitutions have been made.
[0062] The invention includes DIG-3 variants having a core toxin
segment that is 90%, 95% or 97% identical to amino acids 1-643 of
SEQ ID NO:2 or amino acids 73-643 of SEQ ID NO:2.
[0063] Variants may be made by making random mutations or the
variants may be designed. In the case of designed mutants, there is
a high probability of generating variants with similar activity to
the native toxin when amino acid identity is maintained in critical
regions of the toxin which account for biological activity or are
involved in the determination of three-dimensional configuration
which ultimately is responsible for the biological activity. A high
probability of retaining activity will also occur if substitutions
are conservative. Amino acids may be placed in the following
classes: non-polar, uncharged polar, basic, and acidic.
Conservative substitutions whereby an amino acid of one class is
replaced with another amino acid of the same type are least likely
to materially alter the biological activity of the variant. Table 2
provides a listing of examples of amino acids belonging to each
class.
TABLE-US-00002 TABLE 2 Class of Amino Acid Examples of Amino Acids
Nonpolar Side Chains Ala (A), Val (V), Leu (L), Ile (I), Pro (P),
Met (M), Phe (F), Trp (W) Uncharged Polar Side Chains Gly (G), Ser
(S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) Acidic Side Chains
Asp (D), Glu (E) Basic Side Chains Lys (K), Arg (R), His (H)
Beta-branched Side Chains Thr, Val, Ile Aromatic Side Chains Tyr,
Phe, Trp, His
[0064] In some instances, non-conservative substitutions can also
be made. The critical factor is that these substitutions must not
significantly detract from the biological activity of the toxin.
Variants include polypeptides that differ in amino acid sequence
due to mutagenesis. Variant proteins encompassed by the present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, namely,
retaining pesticidal activity.
[0065] Variant proteins can also be designed that differ at the
sequence level but that retain the same or similar overall
essential three-dimensional structure, surface charge distribution,
and the like. See e.g. U.S. Pat. No. 7,058,515; Larson et al.
(2002); Stemmer (1994a, 1994b, 1995); and Crameri et al. (1996a,
1996b, 1997).
[0066] Nucleic Acids.
[0067] Isolated nucleic acids encoding DIG-3 toxins are one aspect
of the present invention. This includes nucleic acids encoding SEQ
ID NO:2 and SEQ ID NO:5, and complements thereof, as well as other
nucleic acids that encode insecticidal variants of SEQ ID NO:2.
Because of the redundancy of the genetic code, a variety of
different DNA sequences can encode the amino acid sequences
disclosed herein. It is well within the skill of a person trained
in the art to create these alternative DNA sequences encoding the
same, or essentially the same, toxins.
[0068] Gene Synthesis.
[0069] DNA sequences encoding the improved Cry proteins described
herein can be made by a variety of methods well-known in the art.
For example, synthetic gene segments and synthetic genes can be
made by phosphite tri-ester and phosphoramidite chemistry
(Caruthers et al., 1987), and commercial vendors are available to
perform DNA synthesis on demand. Sequences encoding full-length
DIG-3 proteins can be assembled in a variety of ways including, for
example, by ligation of restriction fragments or polymerase chain
reaction assembly of overlapping oligonucleotides (Stewart and
Burgin, 2005). Further, sequences encoding terminal deletions can
be made by PCR amplification using site-specific terminal
oligonucleotides.
[0070] Nucleic acids encoding DIG-3 toxins can be made for example,
by synthetic construction by methods currently practiced by any of
several commercial suppliers. (See for example, U.S. Pat. No.
7,482,119 B2). These nucleic acids, or portions or variants
thereof, may also be constructed synthetically, for example, by use
of a gene synthesizer and the design methods of, for example, U.S.
Pat. No. 5,380,831. Alternatively, variations of synthetic or
naturally occurring genes may be readily constructed using standard
molecular biological techniques for making point mutations.
Fragments of these genes can also be made using commercially
available exonucleases or endonucleases according to standard
procedures. For example, enzymes such as Bal31 or site-directed
mutagenesis can be used to systematically cut off nucleotides from
the ends of these genes. Also, gene fragments which encode active
toxin fragments may be obtained using a variety of restriction
enzymes.
[0071] Given the amino acid sequence for a DIG-3 toxin, a coding
sequence can be designed by reverse translating the coding sequence
using codons preferred by the intended host, and then refining the
sequence using alternative codons to remove sequences that might
cause problems and provide periodic stop codons to eliminate long
open coding sequences in the non-coding reading frames.
[0072] Quantifying Sequence Identity.
[0073] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes. The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences (i.e. percent identity=number of
identical positions/total number of positions (e.g. overlapping
positions).times.100). In one embodiment, the two sequences are the
same length. The percent identity between two sequences can be
determined using techniques similar to those described below, with
or without allowing gaps. In calculating percent identity,
typically exact matches are counted.
[0074] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A nonlimiting
example of such an algorithm is that of Karlin and Altschul (1990),
modified as in Karlin and Altschul (1993), and incorporated into
the BLASTN and BLASTX programs. BLAST searches may be conveniently
used to identify sequences homologous (similar) to a query sequence
in nucleic or protein databases. BLASTN searches can be performed,
(score=100, word length=12) to identify nucleotide sequences having
homology to claimed nucleic acid molecules of the invention. BLASTX
searches can be performed (score=50, word length=3) to identify
amino acid sequences having homology to claimed insecticidal
protein molecules of the invention.
[0075] Gapped BLAST (Altschul et al., 1997) can be utilized to
obtain gapped alignments for comparison purposes, Alternatively,
PSI-Blast can be used to perform an iterated search that detects
distant relationships between molecules (Altschul et al., 1997).
When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the
default parameters of the respective programs can be used. See
www.ncbi.nlm.nih.gov.
[0076] A non-limiting example of a mathematical algorithm utilized
for the comparison of sequences is the ClustalW algorithm (Thompson
et al., 1994). ClustalW compares sequences and aligns the entirety
of the amino acid or DNA sequence, and thus can provide data about
the sequence conservation of the entire amino acid sequence or
nucleotide sequence. The ClustalW algorithm is used in several
commercially available DNA/amino acid analysis software packages,
such as the ALIGNX module of the Vector NTI Program Suite
(Invitrogen, Inc., Carlsbad, Calif.). When aligning amino acid
sequences with ALIGNX, one may conveniently use the default
settings with a Gap open penalty of 10, a Gap extend penalty of 0.1
and the blosum63mt2 comparison matrix to assess the percent amino
acid similarity (consensus) or identity between the two sequences.
When aligning DNA sequences with ALIGNX, one may conveniently use
the default settings with a Gap open penalty of 15, a Gap extend
penalty of 6.6 and the swgapdnamt comparison matrix to assess the
percent identity between the two sequences.
[0077] Another non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is that of Myers and
Miller (1988). Such an algorithm is incorporated into the
wSTRETCHER program, which is part of the wEMBOSS sequence alignment
software package (available at http://emboss.sourceforge.net/).
wSTRETCHER calculates an optimal global alignment of two sequences
using a modification of the classic dynamic programming algorithm
which uses linear space. The substitution matrix, gap insertion
penalty and gap extension penalties used to calculate the alignment
may be specified. When utilizing the wSTRETCHER program for
comparing nucleotide sequences, a Gap open penalty of 16 and a Gap
extend penalty of 4 can be used with the scoring matrix file
EDNAFULL. When used for comparing amino acid sequences, a Gap open
penalty of 12 and a Gap extend penalty of 2 can be used with the
EBLOSUM62 scoring matrix file.
[0078] A further non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is that of Needleman and
Wunsch (1970), which is incorporated in the sequence alignment
software packages GAP Version 10 and wNEEDLE
(http://emboss.sourceforge.net/). GAP Version 10 may be used to
determine sequence identity or similarity using the following
parameters: for a nucleotide sequence, % identity and % similarity
are found using GAP Weight of 50 and Length Weight of 3, and the
nwsgapdna. cmp scoring matrix. For amino acid sequence comparison,
% identity or % similarity are determined using GAP weight of 8 and
length weight of 2, and the BLOSUM62 scoring program.
[0079] wNEEDLE reads two input sequences, finds the optimum
alignment (including gaps) along their entire length, and writes
their optimal global sequence alignment to file. The algorithm
explores all possible alignments and chooses the best, using a
scoring matrix that contains values for every possible residue or
nucleotide match. wNEEDLE finds the alignment with the maximum
possible score, where the score of an alignment is equal to the sum
of the matches taken from the scoring matrix, minus penalties
arising from opening and extending gaps in the aligned sequences.
The substitution matrix and gap opening and extension penalties are
user-specified. When amino acid sequences are compared, a default
Gap open penalty of 10, a Gap extend penalty of 0.5, and the
EBLOSUM62 comparison matrix are used. When DNA sequences are
compared using wNEEDLE, a Gap open penalty of 10, a Gap extend
penalty of 0.5, and the EDNAFULL comparison matrix are used.
[0080] Equivalent programs may also be used. By "equivalent
program" is intended any sequence comparison program that, for any
two sequences in question, generates an alignment having identical
nucleotide or amino acid residue matches and an identical percent
sequence identity when compared to the corresponding alignment
generated by ALIGNX, wNEEDLE, or wSTRETCHER. The % identity is the
percentage of identical matches between the two sequences over the
reported aligned region (including any gaps in the length) and the
% similarity is the percentage of matches between the two sequences
over the reported aligned region (including any gaps in the
length).
[0081] Alignment may also be performed manually by inspection.
[0082] Recombinant Hosts.
[0083] The toxin-encoding genes of the subject invention can be
introduced into a wide variety of microbial or plant hosts.
Expression of the toxin gene results, directly or indirectly, in
the intracellular production and maintenance of the pesticidal
protein. With suitable microbial hosts, e.g. Pseudomonas, the
microbes can be applied to the environment of the pest, where they
will proliferate and be ingested. The result is a control of the
pest. Alternatively, the microbe hosting the toxin gene can be
treated under conditions that prolong the activity of the toxin and
stabilize the cell. The treated cell, which retains the toxic
activity, then can be applied to the environment of the target
pest.
[0084] Where the B.t. toxin gene is introduced via a suitable
vector into a microbial host, and said host is applied to the
environment in a living state, it is essential that certain host
microbes be used. Microorganism hosts are selected which are known
to occupy the "phytosphere" (phylloplane, phyllosphere,
rhizosphere, and/or rhizoplane) of one or more crops of interest.
These microorganisms are selected so as to be capable of
successfully competing in the particular environment (crop and
other insect habitats) with the wild-type indigenous
microorganisms, provide for stable maintenance and expression of
the gene expressing the polypeptide pesticide, and, desirably,
provide for improved protection of the pesticide from environmental
degradation and inactivation.
[0085] A large number of microorganisms are known to inhabit the
phylloplane (the surface of the plant leaves) and/or the
rhizosphere (the soil surrounding plant roots) of a wide variety of
important crops. These microorganisms include bacteria, algae, and
fungi. Of particular interest are microorganisms, such as bacteria,
e.g. genera Pseudomonas, Erwinia, Serratia, Klebsiella,
Xanthomonas, Streptomyces, Rhizobium, Sinorhizobium,
Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,
Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and
Alcaligenes; fungi, particularly yeast, e.g. genera Saccharomyces,
Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and
Aureobasidium. Of particular interest are such phytosphere
bacterial species as Pseudomonas syringae, Pseudomonas fluorescens,
Serratia marcescens, Acetobacter xylinum, Agrobacterium
tumefaciens, Agrobacterium radiobacter, Rhodopseudomonas
spheroides, Xanthomonas campestris, Sinorhizobium nieliloti
(formerly Rhizobium meliloti), Alcaligenes eutrophus, and
Azotobacter vinelandii; and phytosphere yeast species such as
Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of
particular interest are the pigmented microorganisms.
Methods of Controlling Insect Pests
[0086] When an insect comes into contact with an effective amount
of toxin delivered via transgenic plant expression, formulated
protein compositions(s), sprayable protein composition(s), a bait
matrix or other delivery system, the results are typically death of
the insect, or the insects do not feed upon the source which makes
the toxins available to the insects.
[0087] The subject protein toxins can be "applied" or provided to
contact the target insects in a variety of ways. For example,
transgenic plants (wherein the protein is produced by and present
in the plant) can be used and are well-known in the art. Expression
of the toxin genes can also be achieved selectively in specific
tissues of the plants, such as the roots, leaves, etc. This can be
accomplished via the use of tissue-specific promoters, for example.
Spray-on applications are another example and are also known in the
art. The subject proteins can be appropriately formulated for the
desired end use, and then sprayed (or otherwise applied) onto the
plant and/or around the plant/to the vicinity of the plant to be
protected--before an infestation is discovered, after target
insects are discovered, both before and after, and the like. Bait
granules, for example, can also be used and are known in the
art.
Transgenic Plants
[0088] The subject proteins can be used to protect practically any
type of plant from damage by a Lepidopteran insect. Nonlimiting
examples of such plants include maize, sunflower, soybean, cotton,
canola, rice, sorghum, wheat, barley, vegetables, ornamentals,
peppers (including hot peppers), sugar beets, fruit, and turf; to
name but a few. Methods for transforming plants are well known in
the art, and illustrative transformation methods are described in
the Examples.
[0089] A preferred embodiment of the subject invention is the
transformation of plants with genes encoding the subject
insecticidal protein or its variants. The transformed plants are
resistant to attack by an insect target pest by virtue of the
presence of controlling amounts of the subject insecticidal protein
or its variants in the cells of the transformed plant. By
incorporating genetic material that encodes the insecticidal
properties of the B.t. insecticidal toxins into the genome of a
plant eaten by a particular insect pest, the adult or larvae would
die after consuming the food plant. Numerous members of the
monocotyledonous and dicotyledonous classifications have been
transformed. Transgenic agronomic crops as well as fruits and
vegetables are of commercial interest. Such crops include, but are
not limited to, maize, rice, soybeans, canola, sunflower, alfalfa,
sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like.
Several techniques exist for introducing foreign genetic material
into plant cells, and for obtaining plants that stably maintain and
express the introduced gene. Such techniques include acceleration
of genetic material coated onto microparticles directly into cells
(U.S. Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may
be transformed using Agrobacterium technology, see U.S. Pat. No.
5,177,010, U.S. Pat. No. 5,104,310, European Patent Application No.
0131624B I, European Patent Application No. 120516, European Patent
Application No. 159418B1, European Patent Application No. 176112,
U.S. Pat. No. 5,149,645, U.S. Pat. No. 5,469,976, U.S. Pat. No.
5,464,763, U.S. Pat. No. 4,940,838, U.S. Pat. No. 4,693,976,
European Patent Application No. 116718, European Patent Application
No. 290799, European Patent Application No. 320500, European Patent
Application No. 604662, European Patent Application No. 627752,
European Patent Application No. 0267159, European Patent
Application No. 0292435, U.S. Pat. No. 5,231,019, U.S. Pat. No.
5,463,174, U.S. Pat. No. 4,762,785, U.S. Pat. No. 5,004,863, and
U.S. Pat. No. 5,159,135. Other transformation technology includes
WHISKERS.TM. technology, see U.S. Pat. No. 5,302,523 and U.S. Pat.
No. 5,464,765. Electroporation technology has also been used to
transform plants, see WO 87/06614, U.S. Pat. No. 5,472,869, U.S.
Pat. No. 5,384,253, WO 9209696, and WO 9321335. All of these
transformation patents and publications are incorporated by
reference. In addition to numerous technologies for transforming
plants, the type of tissue which is contacted with the foreign
genes may vary as well. Such tissue would include but would not be
limited to embryogenic tissue, callus tissue types I and II,
hypocotyl, meristem, and the like. Almost all plant tissues may be
transformed during dedifferentiation using appropriate techniques
within the skill of an artisan.
[0090] Genes encoding DIG-3 toxins can be inserted into plant cells
using a variety of techniques which are well known in the art as
disclosed above. For example, a large number of cloning vectors
comprising a marker that permits selection of the transformed
microbial cells and a replication system functional in Escherichia
coli are available for preparation and modification of foreign
genes for insertion into higher plants. Such manipulations may
include, for example, the insertion of mutations, truncations,
additions, or substitutions as desired for the intended use. The
vectors comprise, for example, pBR322, pUC series, M13 mp series,
pACYC184, etc. Accordingly, the sequence encoding the Cry protein
or variants can be inserted into the vector at a suitable
restriction site. The resulting plasmid is used for transformation
of cells of E. coli, the cells of which are cultivated in a
suitable nutrient medium, then harvested and lysed so that workable
quantities of the plasmid are recovered. Sequence analysis,
restriction fragment analysis, electrophoresis, and other
biochemical-molecular biological methods are generally carried out
as methods of analysis. After each manipulation, the DNA sequence
used can be cleaved and joined to the next DNA sequence. Each
manipulated DNA sequence can be cloned in the same or other
plasmids.
[0091] The use of T-DNA-containing vectors for the transformation
of plant cells has been intensively researched and sufficiently
described in EP 120516; Lee and Gelvin (2008), Fraley et al.
(1986), and An et al. (1985), and is well established in the
field.
[0092] Once the inserted DNA has been integrated into the plant
genome, it is relatively stable throughout subsequent generations.
The vector used to transform the plant cell normally contains a
selectable marker gene encoding a protein that confers on the
transformed plant cells resistance to a herbicide or an antibiotic,
such as bialaphos, kanamycin, G418, bleomycin, or hygromycin, inter
alia. The individually employed selectable marker gene should
accordingly permit the selection of transformed cells while the
growth of cells that do not contain the inserted DNA is suppressed
by the selective compound.
[0093] A large number of techniques are available for inserting DNA
into a host plant cell. Those techniques include transformation
with T-DNA delivered by Agrobacterium tumefaciens or Agrobacterium
rhizogenes as the transformation agent. Additionally, fusion of
plant protoplasts with liposomes containing the DNA to be
delivered, direct injection of the DNA, biolistics transformation
(microparticle bombardment), or electroporation, as well as other
possible methods, may be employed.
[0094] In a preferred embodiment of the subject invention, plants
will be transformed with genes wherein the codon usage of the
protein coding region has been optimized for plants. See, for
example, U.S. Pat. No. 5,380,831, which is hereby incorporated by
reference. Also, advantageously, plants encoding a truncated toxin
will be used. The truncated toxin typically will encode about 55%
to about 80% of the full length toxin. Methods for creating
synthetic B.t. genes for use in plants are known in the art
(Stewart, 2007).
[0095] Regardless of transformation technique, the gene is
preferably incorporated into a gene transfer vector adapted to
express the B.t insecticidal toxin genes and variants in the plant
cell by including in the vector a plant promoter. In addition to
plant promoters, promoters from a variety of sources can be used
efficiently in plant cells to express foreign genes. For example,
one may use promoters of bacterial origin, such as the octopine
synthase promoter, the nopaline synthase promoter, and the
mannopine synthase promoter. Promoters of plant virus origin may be
used, for example, the 35S and 19S promoters of Cauliflower Mosaic
Virus, a promoter from Cassava Vein Mosaic Virus, and the like.
Plant promoters include, but are not limited to,
ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),
beta-conglycinin promoter, phaseolin promoter, ADH (alcohol
dehydrogenase) promoter, heat-shock promoters, ADF (actin
depolymerization factor) promoter, ubiquitin promoter, actin
promoter, and tissue specific promoters. Promoters may also contain
certain enhancer sequence elements that may improve the
transcription efficiency. Typical enhancers include but are not
limited to ADH1-intron 1 and ADH1-intron 6. Constitutive promoters
may be used. Constitutive promoters direct continuous gene
expression in nearly all cells types and at nearly all times (e.g.,
actin, ubiquitin, CaMV 35S). Tissue specific promoters are
responsible for gene expression in specific cell or tissue types,
such as the leaves or seeds (e.g. zein, oleosin, napin, ACP (Acyl
Carrier Protein) promoters), and these promoters may also be used.
Promoters may also be used that are active during a certain stage
of the plants' development as well as active in specific plant
tissues and organs. Examples of such promoters include but are not
limited to promoters that are root specific, pollen-specific,
embryo specific, corn silk specific, cotton fiber specific, seed
endosperm specific, phloem specific, and the like.
[0096] Under certain circumstances it may be desirable to use an
inducible promoter. An inducible promoter is responsible for
expression of genes in response to a specific signal, such as:
physical stimulus (e.g. heat shock genes); light (e.g. RUBP
carboxylase); hormone (e.g. glucocorticoid); antibiotic (e.g.
tetracycline); metabolites; and stress (e.g. drought). Other
desirable transcription and translation elements that function in
plants may be used, such as 5' untranslated leader sequences, RNA
transcription termination sequences and poly-adenylate addition
signal sequences. Numerous plant-specific gene transfer vectors are
known to the art.
[0097] Transgenic crops containing insect resistance (IR) traits
are prevalent in corn and cotton plants throughout North America,
and usage of these traits is expanding globally. Commercial
transgenic crops combining IR and herbicide tolerance (HT) traits
have been developed by multiple seed companies. These include
combinations of IR traits conferred by B.t. insecticidal proteins
and HT traits such as tolerance to Acetolactate Synthase (ALS)
inhibitors such as sulfonylureas, imidazolinones,
triazolopyrimidine, sulfonanilides, and the like, Glutamine
Synthetase (GS) inhibitors such as bialaphos, glufosinate, and the
like, 4-HydroxyPhenylPyruvate Dioxygenase (HPPD) inhibitors such as
mesotrione, isoxaflutole, and the like,
5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such
as glyphosate and the like, and Acetyl-Coenzyme A Carboxylase
(ACCase) inhibitors such as haloxyfop, quizalofop, diclofop, and
the like. Other examples are known in which transgenically provided
proteins provide plant tolerance to herbicide chemical classes such
as phenoxy acids herbicides and pyridyloxyacetates auxin herbicides
(see WO 2007/053482 A2), or phenoxy acids herbicides and
aryloxyphenoxypropionates herbicides (see WO 2005107437 A2, A3).
The ability to control multiple pest problems through IR traits is
a valuable commercial product concept, and the convenience of this
product concept is enhanced if insect control traits and weed
control traits are combined in the same plant. Further, improved
value may be obtained via single plant combinations of IR traits
conferred by a B.t. insecticidal protein such as that of the
subject invention, with one or more additional HT traits such as
those mentioned above, plus one or more additional input traits
(e.g. other insect resistance conferred by B.t.-derived or other
insecticidal proteins, insect resistance conferred by mechanisms
such as RNAi and the like, nematode resistance, disease resistance,
stress tolerance, improved nitrogen utilization, and the like), or
output traits (e.g. high oils content, healthy oil composition,
nutritional improvement, and the like). Such combinations may be
obtained either through conventional breeding (breeding stack) or
jointly as a novel transformation event involving the simultaneous
introduction of multiple genes (molecular stack). Benefits include
the ability to manage insect pests and improved weed control in a
crop plant that provides secondary benefits to the producer and/or
the consumer. Thus, the subject invention can be used in
combination with other traits to provide a complete agronomic
package of improved crop quality with the ability to flexibly and
cost effectively control any number of agronomic issues.
Target Pests
[0098] The DIG-3 toxins of the invention are particularly suitable
for use in control of Lepidopteran insects. Lepidopterans are an
important group of agricultural, horticultural, and household pests
which cause a very large amount of damage each year. This insect
order encompasses foliar- and root-feeding larvae and adults.
Lepidopteran insect pests include, but are not limited to: Achoroia
grisella, Acleris gloverana, Acleris variana, Adoxophyes orana,
Agrotis ipsilon (black cutworm), Alabama argillacea, Alsophila
pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia
lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia
gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx
mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp.,
Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia
latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus
sibericus, Desmia feneralis, Diaphania hyalinata, Diaphania
nitidalis, Diatraea grandiosella (southwestern corn borer),
Diatraea saccharalis (sugarcane borer), Ennomos subsignaria,
Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene
acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia
ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria
mellonella, Grapholita molesta, Harrisina americana, Helicoverpa
subflexa, Helicoverpa zea (corn earworm), Heliothis virescens
(tobacco budworm), Hemileuca oliviae, Homoeosoma electellum
(sunflower head moth), Hyphantia cunea, Keiferia lycopersicella,
Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa,
Leucoma salicis, Lobesia botrana, Loxagrotis albicosta (western
bean cutworm), Loxostege sticticalis, Lymantria dispar, Macalla
thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra
configurata (bertha armyworm), Manduca quinquemaculata, Manduca
sexta (tobacco hornworm), Maruca testulalis, Melanchra picta,
Operophtera brumata, Orgyia sp., Ostrinia nubilalis (European corn
borer), Paleacrita vernata, Papiapema nebris (common stalk borer),
Papilio cresphontes, Pectinophora gossypiella, Phryganidia
califormica, Phyllonorycter blancardella, Pieris napi, Pieris
rapae, Plathypena scabra, Platynota flouendana, Platynota stultana,
Platyptilia carduidactyla, Plodia interpunctella, Plutella
xylostella (diamondback moth), Pontia protodice, Pseudaletia
unipuncta (armyworm), Pseudoplasia includens, Rachiplusia nu
(Argentine looper), Sabulodes aegrotata, Schizura concinna,
Sitotroga cerealella, Spilonta ocellana, Spodoptera frugiperda
(fall armyworm), Spodoptera exigua (beet armyworm), Thaurnstopoea
pityocampa, Ensola bisselliella, Trichoplusia hi, Udea rubigalis,
Xylomyges curiails, and Yponomeuta padella.
[0099] Use of DIG-3 toxins to control Coleopteran pests of crop
plants is also contemplated. In some embodiments, Cry proteins may
be economically deployed for control of insect pests that include
but are not limited to, for example, rootworms such as Diabrotica
undecimpunctata howardi (southern corn rootworm), Diabrotica
longicornis barberi (northern corn rootworm), and Diabrotica
virgifera (western corn rootworm), and grubs such as the larvae of
Cyclocephala borealis (northern masked chafer), Cyclocephala
immaculate (southern masked chafer), and Popillia japonica
(Japanese beetle).
[0100] Use of the DIG-3 toxins to control parasitic nematodes
including, but not limited to, root knot nematode (Meloidogyne
icognita) and soybean cyst nematode (Heterodera glycines) is also
contemplated.
Antibody Detection of DIG-3 Toxins
[0101] Anti-toxin antibodies. Antibodies to the toxins disclosed
herein, or to equivalent toxins, or fragments of these toxins, can
readily be prepared using standard procedures in this art. Such
antibodies are useful to detect the presence of the DIG-3
toxins.
[0102] Once the B.t. insecticidal toxin has been isolated,
antibodies specific for the toxin may be raised by conventional
methods that are well known in the art. Repeated injections into a
host of choice over a period of weeks or months will elicit an
immune response and result in significant anti-B.t toxin serum
titers. Preferred hosts are mammalian species and more highly
preferred species are rabbits, goats, sheep and mice. Blood drawn
from such immunized animals may be processed by established methods
to obtain antiserum (polyclonal antibodies) reactive with the B.t.
insecticidal toxin. The antiserum may then be affinity purified by
adsorption to the toxin according to techniques known in the art.
Affinity purified antiserum may be further purified by isolating
the immunoglobulin fraction within the antiserum using procedures
known in the art. The resulting material will be a heterogeneous
population of immunoglobulins reactive with the B.t. insecticidal
toxin.
[0103] Anti-B.t. toxin antibodies may also be generated by
preparing a semi-synthetic immunogen consisting of a synthetic
peptide fragment of the B.t. insecticidal toxin conjugated to an
immunogenic carrier. Numerous schemes and instruments useful for
making peptide fragments are well known in the art. Many suitable
immunogenic carriers such as bovine serum albumin or keyhole limpet
hemocyanin are also well known in the art, as are techniques for
coupling the immunogen and carrier proteins. Once the
semi-synthetic immunogen has been constructed, the procedure for
making antibodies specific for the B.t. insecticidal toxin fragment
is identical to those used for making antibodies reactive with
natural B.t. toxin.
[0104] Anti-B.t. toxin monoclonal antibodies (MAbs) are readily
prepared using purified B.t. insecticidal toxin. Methods for
producing MAbs have been practiced for over 20 years and are well
known to those of ordinary skill in the art. Repeated
intraperitoneal or subcutaneous injections of purified B.t.
insecticidal toxin in adjuvant will elicit an immune response in
most animals. Hyperimmunized B-lymphocytes are removed from the
animal and fused with a suitable fusion partner cell line capable
of being cultured indefinitely. Preferred animals whose
B-lymphocytes may be hyperimmunized and used in the production of
MAbs are mammals. More preferred animals are rats and mice and most
preferred is the BALB/c mouse strain.
[0105] Numerous mammalian cell lines are suitable fusion partners
for the production of hybridomas. Many such lines are available
from the American Type Culture Collection (ATCC, Manassas, Va.) and
commercial suppliers. Preferred fusion partner cell lines are
derived from mouse myelomas and the HL-1.RTM. Friendly myeloma-653
cell line (Ventrex, Portland, Me.) is most preferred. Once fused,
the resulting hybridomas are cultured in a selective growth medium
for one to two weeks. Two well known selection systems are
available for eliminating unfused myeloma cells, or fusions between
myeloma cells, from the mixed hybridoma culture. The choice of
selection system depends on the strain of mouse immunized and
myeloma fusion partner used. The AAT selection system, described by
Taggart and Samloff, (1983), may be used; however, the HAT
(hypoxanthine, aminopterin, thymidine) selection system, described
by Littlefield, (1964), is preferred because of its compatibility
with the preferred mouse strain and fusion partner mentioned above.
Spent growth medium is screened for immunospecific MAb secretion.
Enzyme linked immunosorbent assay (ELISA) procedures are best
suited for this purpose; though, radioimmunoassays adapted for
large volume screening are also acceptable. Multiple screens
designed to consecutively pare down the considerable number of
irrelevant or less desired cultures may be performed. Cultures that
secrete MAbs reactive with the B.t. insecticidal toxin may be
screened for cross-reactivity with known B.t. insecticidal toxins.
MAbs that preferentially bind to the preferred B.t. insecticidal
toxin may be isotyped using commercially available assays.
Preferred MAbs are of the IgG class, and more highly preferred MAbs
are of the IgG.sub.1 and IgG.sub.2a subisotypes.
[0106] Hybridoma cultures that secrete the preferred MAbs may be
sub-cloned several times to establish monoclonality and stability.
Well known methods for sub-cloning eukaryotic, non-adherent cell
cultures include limiting dilution, soft agarose and fluorescence
activated cell sorting techniques. After each subcloning, the
resultant cultures preferably are re-assayed for antibody secretion
and isotype to ensure that a stable preferred MAb-secreting culture
has been established.
[0107] The anti-B.t. toxin antibodies are useful in various methods
of detecting the claimed B.t. insecticidal toxin of the instant
invention, and variants or fragments thereof. It is well known that
antibodies labeled with a reporting group can be used to identify
the presence of antigens in a variety of milieus. Antibodies
labeled with radioisotopes have been used for decades in
radioimmunoassays to identify, with great precision and
sensitivity, the presence of antigens in a variety of biological
fluids. More recently, enzyme labeled antibodies have been used as
a substitute for radiolabeled antibodies in the ELISA assay.
Further, antibodies immunoreactive to the B.t. insecticidal toxin
of the present invention can be bound to an immobilizing substance
such as a polystyrene well or particle and used in immunoassays to
determine whether the B.t. toxin is present in a test sample.
Detection Using Nucleic Acid Probes
[0108] A further method for identifying the toxins and genes of the
subject invention is through the use of oligonucleotide probes.
These probes are detectable nucleotide sequences. These sequences
may be rendered detectable by virtue of an appropriate radioactive
label or may be made inherently fluorescent as described in, for
example, U.S. Pat. No. 6,268,132. As is well known in the art, if
the probe molecule and nucleic acid sample hybridize by forming
strong base-pairing bonds between the two molecules, it can be
reasonably assumed that the probe and sample have substantial
sequence homology. Preferably, hybridization is conducted under
stringent conditions by techniques well-known in the art, as
described, for example, in Keller and Manak (1993). Detection of
the probe provides a means for determining in a known manner
whether hybridization has occurred. Such a probe analysis provides
a rapid method for identifying toxin-encoding genes of the subject
invention. The nucleotide segments which are used as probes
according to the invention can be synthesized using a DNA
synthesizer and standard procedures. These nucleotide sequences can
also be used as PCR primers to amplify genes of the subject
invention.
Nucleic Acid Hybridization
[0109] As is well known to those skilled in molecular biology,
similarity of two nucleic acids can be characterized by their
tendency to hybridize. As used herein the terms "stringent
conditions" or "stringent hybridization conditions" are intended to
refer to conditions under which a probe will hybridize (anneal) to
its target sequence to a detectably greater degree than to other
sequences (e.g. at least 2-fold over background). Stringent
conditions are sequence-dependent and will be different in
different circumstances. By controlling the stringency of the
hybridization and/or washing conditions, target sequences that are
100% complementary to the probe can be identified (homologous
probing). Alternatively, stringency conditions can be adjusted to
allow some mismatching in sequences so that lower degrees of
similarity are detected (heterologous probing). Generally, a probe
is less than about 1000 nucleotides in length, preferably less than
500 nucleotides in length.
[0110] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to pH
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g. 10 to 50 nucleotides) and at least about 60.degree. C.
for long probes (e.g. greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide Exemplary low stringency conditions
include hybridization with a buffer solution of 30% to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37.degree.
C. and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50.degree. C. to 55.degree. C.
Exemplary moderate stringency conditions include hybridization in
40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37.degree. C. and a
wash in 0.5.times. to 1.times.SSC at 55.degree. C. to 60.degree. C.
Exemplary high stringency conditions include hybridization in 50%
formamide, 1 M NaCl, 1% SDS at 37.degree. C. and a wash in
0.1.times.SSC at 60.degree. C. to 65.degree. C. Optionally, wash
buffers may comprise about 0.1% to about 1% SDS. Duration of
hybridization is generally less than about 24 hours, usually about
4 to about 12 hours.
[0111] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA/DNA hybrids, the
thermal melting point (T.sub.m) is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, hybridization conditions, and/or wash conditions can be
adjusted to facilitate annealing of sequences of the desired
identity. For example, if sequences with >90% identity are
sought, the T.sub.m can be decreased 10.degree. C. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the T.sub.n, for the specific sequence and its complement at a
defined ionic strength and pH. However, highly stringent conditions
can utilize a hybridization and/or wash at 1.degree. C., 2.degree.
C., 3.degree. C., or 4.degree. C. lower than the T.sub.m;
moderately stringent conditions can utilize a hybridization and/or
wash at 6.degree. C., 7.degree. C., 8.degree. C., 9.degree. C., or
10.degree. C. lower than the T.sub.m, and low stringency conditions
can utilize a hybridization and/or wash at 11.degree. C.,
12.degree. C., 13.degree. C., 14.degree. C., 15.degree. C., or
20.degree. C. lower than the T.sub.m.
[0112] T.sub.m (in .degree. C.) may be experimentally determined or
may be approximated by calculation. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl
(1984):
T.sub.m(.degree. C.)=81.5.degree. C.+16.6(log M)+0.41(% GC)-0.61(%
formamide)-500/L;
where M is the molarity of monovalent cations, % GC is the
percentage of guanosine and cytosine nucleotides in the DNA, %
formamide is the percentage of formamide in the hybridization
solution, and L is the length of the hybrid in base pairs.
[0113] Alternatively, the T.sub.m is described by the following
formula (Beltz et al., 1983).
T.sub.m(.degree. C.)=81.5.degree. C.+16.6(log [Na.sup.+])+0.41(%
GC)-0.61(% formamide)-600/L
where [Na.sup.+] is the molarity of sodium ions, % GC is the
percentage of guanosine and cytosine nucleotides in the DNA, %
formamide is the percentage of formamide in the hybridization
solution, and L is the length of the hybrid in base pairs.
[0114] Using the equations, hybridization and wash compositions,
and desired T.sub.m, those of ordinary skill will understand that
variations in the stringency of hybridization and/or wash solutions
are inherently described. If the desired degree of mismatching
results in a T.sub.m of less than 45.degree. C. (aqueous solution)
or 32.degree. C. (formamide solution), it is preferred to increase
the SSC concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen (1993) and Ausubel et al. (1995) Also see Sambrook et al.
(1989).
[0115] Hybridization of immobilized DNA on Southern blots with
radioactively labeled gene-specific probes may be performed by
standard methods Sambrook et al., supra.). Radioactive isotopes
used for labeling polynucleotide probes may include 32P, 33P, 14C,
or 3H. Incorporation of radioactive isotopes into polynucleotide
probe molecules may be done by any of several methods well known to
those skilled in the field of molecular biology. (See, e.g.
Sambrook et al., supra.) In general, hybridization and subsequent
washes may be carried out under stringent conditions that allow for
detection of target sequences with homology to the claimed toxin
encoding genes. For double-stranded DNA gene probes, hybridization
may be carried out overnight at 20.degree.-25.degree. C. below the
T.sub.m of the DNA hybrid in 6.times.SSPE, 5.times.Denhardt's
Solution, 0.1% SDS, 0.1 mg/mL denatured DNA [20.times.SSPE is 3M
NaCl, 0.2 M NaHPO.sub.4, and 0.02M EDTA (ethylenediamine
tetra-acetic acid sodium salt); 100.times.Denhardt's Solution is 20
gm/L Polyvinylpyrollidone, 20 gm/L Ficoll type 400 and 20 gm/L
Bovine Serum Albumin (fraction V)].
[0116] Washes may typically be carried out as follows: [0117] Twice
at room temperature for 15 minutes in lx SSPE, 0.1% SDS (low
stringency wash). [0118] Once at T.sub.m-20.degree. C. for 15
minutes in 0.2.times.SSPE, 0.1% SDS (moderate stringency wash).
[0119] For oligonucleotide probes, hybridization may be carried out
overnight at 10.degree.-20.degree. C. below the T.sub.m of the
hybrid in 6.times.SSPE, 5.times.Denhardt's solution, 0.1% SDS, 0.1
mg/mL denatured DNA. T.sub.m for oligonucleotide probes may be
determined by the following formula (Suggs et al., 1981).
T.sub.m(.degree. C.)=2(number of T/A base pairs)+4(number of G/C
base pairs)
[0120] Washes may typically be carried out as follows: [0121] Twice
at room temperature for 15 minutes 1.times.SSPE, 0.1% SDS (low
stringency wash). [0122] Once at the hybridization temperature for
15 minutes in 1.times.SSPE, 0.1% SDS (moderate stringency
wash).
[0123] Probe molecules for hybridization and hybrid molecules
formed between probe and target molecules may be rendered
detectable by means other than radioactive labeling. Such alternate
methods are intended to be within the scope of this invention.
[0124] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety to the extent they are not inconsistent
with the explicit teachings of this specification. Unless
specifically indicated or implied, the terms "a", "an", and "the"
signify "at least one" as used herein. By the use of the term
"genetic material" herein, it is meant to include all genes,
nucleic acid, DNA and RNA.
[0125] For designations of nucleotide residues of polynucleotides,
DNA, RNA, oligonucleotides, and primers, and for designations of
amino acid residues of proteins, standard IUPAC abbreviations are
employed throughout this document. Nucleic acid sequences are
presented in the standard 5' to 3' direction, and protein sequences
are presented in the standard amino (N) terminal to carboxy (C)
terminal direction.
[0126] Following are examples that illustrate procedures for
practicing the invention. It should be understood that the examples
and embodiments described herein are for illustrative purposes only
and that various modifications or changes in light thereof will be
suggested to persons skilled in the art and are to be included
within the spirit and purview of this application and the scope of
the appended claims. These examples should not be construed as
limiting All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted. All temperatures
are in degrees Celsius.
Example 1
Isolation of a Gene Encoding DIG-3 Toxin
[0127] Nucleic acid encoding the insecticidal Cry protein
designated herein as DIG-3 was isolated from genomic DNA of B.t.
strain PS46L by PCR using a degenerate forward primer that
hybridized to bases 1286 to 1311 of SEQ ID NO:1, and a mismatched
reverse primer that hybridized to the complement of bases 2480 to
2499 of SEQ ID NO:1. This pair of primers was used to amplify a
fragment of 1214 bp, corresponding to nucleotides 1286 to 2499 of
SEQ ID NO:1. This sequence was used as the anchor point to begin
genome walking using methods adapted from the GenomeWalker.TM.
Universal Kit (Clontech, Palo Alto, Calif.). The nucleic acid
sequence of a fragment spanning the DIG-3 coding region was
determined SEQ ID NO:1 is the 3771 bp nucleotide sequence encoding
the full length DIG-3 protein. SEQ ID NO:2 is the amino acid
sequence of the full length DIG-3 protein deduced from SEQ ID NO:1.
It is noted that in Bacillus species, protein coding regions such
as that of SEQ ID NO: 1 may initiate with the TTG codon, which
translationally represents the amino acid methionine.
Example 2
Deletion of Domain I .alpha.-Helices from DIG-3
[0128] To improve the insecticidal properties of the DIG-3 toxin,
serial, step-wise deletions are made, each of which removes part of
the N-terminus of the DIG-3 protein. The deletions remove part or
all of .alpha.-helix 1 and part or all of .alpha.-helix 2 in Domain
I, while maintaining the structural integrity of .alpha.-helix 3
through .alpha.-helix 7.
[0129] Deletions were designed as follows. This example utilizes
the full length chimeric DNA sequence encoding the full-length
DIG-3 protein e.g. SEQ ID NO:1 and SEQ ID NO:2, respectively) to
illustrate the design principles with 67 specific variants. It
utilizes the chimeric sequence of SEQ ID NO:5 (DNA encoding DIG-3
core toxin segment fused to Cry1Ab protoxin segment) to provide an
additional 67 specific variants. One skilled in the art will
realize that other DNA sequences encoding all or an N-terminal
portion of the DIG-3 protein may be similarly manipulated to
achieve the desired result. To devise the first deleted variant
coding sequence, all of the bases that encode .alpha.-helix 1 up to
the codon for the proline residue near the beginning of
.alpha.-helix 2A (i.e. P73 for the full length DIG-3 protein of SEQ
ID NO:2), are removed. Thus, elimination of bases 1 to 216 of SEQ
ID NO:1 removes the coding sequence for amino acids 1 to 72 of SEQ
ID NO:2. Reintroduction of a translation initiating ATG
(methionine) codon at the beginning (i.e. in front of the codon
corresponding to amino acid 73 of the full length protein) provides
for the deleted variant coding sequence comprising an open reading
frame of 3555 bases which encodes a deleted variant DIG-3 protein
comprising 1185 amino acids (i.e. methionine plus amino acids 73 to
1256 of the full-length DIG-3 protein). Serial, stepwise deletions
that remove additional codons for a single amino acid corresponding
to residues 73 to 112 of the full-length DIG-3 protein of SEQ ID
NO:2 provide variants lacking part or all of .alpha.-helix 2A and
.alpha.-helix 2B. Thus a second designed deleted variant coding
sequence requires elimination of bases 1 to 219 of SEQ ID NO:1,
thereby removing the coding sequence for amino acids 1-73.
Restoration of a functional open reading frame is again
accomplished by reintroduction of a translation initiation
methionine codon at the beginning of the remaining coding sequence,
thus providing for a second deleted variant coding sequence having
an open reading frame of 3552 bases encoding a deleted variant
DIG-3 protein comprising 1184 amino acids (i.e. methionine plus
amino acids 74 to 1256 of the full-length DIG-3 protein). The last
designed deleted variant coding sequence requires removal of bases
1 to 336 of SEQ ID NO:1, thus eliminating the coding sequence for
amino acids 1 to 112, and, after reintroduction of a translation
initiation methionine codon, providing a deletion variant coding
sequence having an open reading frame of 3435 bases which encodes a
deletion variant DIG-3 protein of 1145 amino acids (i.e. methionine
plus amino acids 113 to 1256 of the full-length DIG-3 protein). As
exemplified, after elimination of the deletion sequence, an
initiator methionine codon is added to the beginning of the
remaining coding sequence to restore a functional open reading
frame. Also as described, an additional glycine codon is to be
added between the methionine codon and the codon for the
instability-determining amino acid in the instance that removal of
the deleted sequence leaves exposed at the N-terminus of the
remaining portion of the full-length protein one of the
instability-determining amino acids as provided above.
[0130] Table 3 describes specific variants designed in accordance
with the strategy described above.
TABLE-US-00003 TABLE 3 Deletion variant protein sequences of the
full-length DIG-3 protein of SEQ ID NO: 2 and the fusion protein
sequence of SEQ ID NO: 5. DIG-3 Residues Deletion added at Variant
NH2 terminus Residues of SEQ ID NO: 2 1 M 73-1256 2 MG 73-1256 3 M
74-1256 4 MG 74-1256 5 M 75-1256 6 M 76-1256 7 M 77-1256 8 M
78-1256 9 MG 78-1256 10 M 79-1256 11 M 80-1256 12 M 81-1256 13 MG
81-1256 14 M 82-1256 15 MG 82-1256 16 M 83-1256 17 M 84-1256 18 MG
84-1256 19 M 85-1256 20 MG 85-1256 21 M 86-1256 22 M 87-1256 23 M
88-1256 24 M 89-1256 25 MG 89-1256 26 M 90-1256 27 MG 90-1256 28 M
91-1256 29 MG 91-1256 30 M 92-1256 31 M 93-1256 32 M 94-1256 33 M
95-1256 34 MG 95-1256 35 M 96-1256 36 MG 96-1256 37 M 97-1256 38 MG
97-1256 39 M 98-1256 40 MG 98-1256 41 M 99-1256 42 MG 99-1256 43 M
100-1256 44 MG 100-1256 45 M 101-1256 46 MG 101-1256 47 M 102-1256
48 MG 102-1256 49 M 103-1256 50 MG 103-1256 51 M 104-1256 52 M
105-1256 53 MG 105-1256 54 M 106-1256 55 MG 106-1256 56 M 107-1256
57 MG 107-1256 58 M 108-1256 59 MG 108-1256 60 M 109-1256 61 MG
109-1256 62 M 110-1256 63 MG 110-1256 64 M 111-1256 65 MG 111-1256
66 M 112-1356 67 M 113-1256 Residues of SEQ ID NO: 5 68 M 73-1188
69 MG 73-1188 70 M 74-1188 71 MG 74-1188 72 M 75-1188 73 M 76-1188
74 M 77-1188 75 M 78-1188 76 MG 78-1188 77 M 79-1188 78 M 80-1188
79 M 81-1188 80 MG 81-1188 81 M 82-1188 82 MG 82-1188 83 M 83-1188
84 M 84-1188 85 MG 84-1188 86 M 85-1188 87 MG 85-1188 88 M 86-1188
89 M 87-1188 90 M 88-1188 91 M 89-1188 92 MG 89-1188 93 M 90-1188
94 MG 90-1188 95 M 91-1188 96 MG 91-1188 97 M 92-1188 98 M 93-1188
99 M 94-1188 100 M 95-1188 101 MG 95-1188 102 M 96-1188 103 MG
96-1188 104 M 97-1188 105 MG 97-1188 106 M 98-1188 107 MG 98-1188
108 M 99-1188 109 MG 99-1188 110 M 100-1188 111 MG 100-1188 112 M
101-1188 113 MG 101-1188 114 M 102-1188 115 MG 102-1188 116 M
103-1188 117 MG 103-1188 118 M 104-1188 119 M 105-1188 120 MG
105-1188 121 M 106-1188 122 MG 106-1188 123 M 107-1188 124 MG
107-1188 125 M 108-1188 126 MG 108-1188 127 M 109-1188 128 MG
109-1188 129 M 110-1188 130 MG 110-1188 131 M 111-1188 132 MG
111-1188 133 M 112-1356 134 M 113-1188
[0131] Nucleic acids encoding the toxins described in Table 3 are
designed in accordance with the general principles for synthetic
genes intended for expression in plants, as discussed above.
Example 3
Design of a Plant-Optimized Version of the Coding Sequence for the
DIG-3 B.t Insecticidal Protein
[0132] A DNA sequence having a plant codon bias was designed and
synthesized to produce the DIG-3 protein in transgenic monocot and
dicot plants. A codon usage table for maize (Zea mays L.) was
calculated from 706 protein coding sequences (CDs) obtained from
sequences deposited in GenBank. Codon usage tables for tobacco
(Nicotiana tabacum, 1268 CDs), canola (Brassica napus, 530 CDs),
cotton (Gossypium hirsutum, 197 CDs), and soybean (Glycine max; ca.
1000 CDs) were downloaded from data at the website
http://www.kazusa.or.jp/codon/. A biased codon set that comprises
highly used codons common to both maize and dicot datasets, in
appropriate weighted average relative amounts, was calculated after
omitting any redundant codon used less than about 10% of total
codon uses for that amino acid in either plant type. To derive a
plant optimized sequence encoding the DIG-3 protein, codon
substitutions to the experimentally determined DIG-3 DNA sequence
were made such that the resulting DNA sequence had the overall
codon composition of the plant-optimized codon bias table. Further
refinements of the sequence were made to eliminate undesirable
restriction enzyme recognition sites, potential plant intron splice
sites, long runs of A/T or C/G residues, and other motifs that
might interfere with RNA stability, transcription, or translation
of the coding region in plant cells. Other changes were made to
introduce desired restriction enzyme recognition sites, and to
eliminate long internal Open Reading Frames (frames other than +1).
These changes were all made within the constraints of retaining the
plant-biased codon composition. Synthesis of the designed sequence
was performed by a commercial vendor (DNA2.0, Menlo Park,
Calif.).
[0133] Additional guidance regarding the production of synthetic
genes can be found in, for example, WO 97/13402 and U.S. Pat. No.
5,380,831.
[0134] A plant-optimized DNA sequence encoding the full length
DIG-3 toxin is given in SEQ ID NO:3. A dicot-optimized DNA sequence
encoding the Cry1Ab protoxin segment is disclosed as SEQ ID NO:6. A
maize-optimized DNA sequence encoding the Cry1Ab protoxin segment
is disclosed as SEQ ID NO:7.
Example 4
Construction of Expression Plasmids Encoding DIG-3 Insecticidal
Toxin and Expression in Bacterial Hosts
[0135] Standard cloning methods were used in the construction of
Pseudomonas fluorescens (pf) expression plasmids engineered to
produce full-length DIG-3 proteins encoded by plant-optimized
coding regions. Restriction endonucleases were obtained from New
England BioLabs (NEB; Ipswich, Mass.) and T4 DNA Ligase
(Invitrogen) was used for DNA ligation. Plasmid preparations were
performed using the NucleoBond.RTM. Xtra Kit (Macherey-Nagel Inc,
Bethlehem, Pa.) or the Plasmid Midi Kit.RTM. (Qiagen), following
the instructions of the suppliers. DNA fragments were purified
using the Millipore Ultrafree.RTM.-DA cartridge (Billerica, Mass.)
after agarose Tris-acetate gel electrophoresis.
[0136] The basic cloning strategy entailed subcloning the DIG-3
toxin coding sequence (CDS) into pDOW1169 at the SpeI and XhoI
restriction sites, whereby it is placed under the expression
control of the Ptac promoter and the rrnBT1T2 terminator from
plasmid pKK223-3 (PL Pharmacia, Milwaukee, Wis.). pDOW1169 is a
medium copy plasmid with the RSF1010 origin of replication, a pyrF
gene, and a ribosome binding site preceding the restriction enzyme
recognition sites into which DNA fragments containing protein
coding regions may be introduced, (US Application 20080193974). The
expression plasmid, designated pDAB4171, was transformed by
electroporation into DC454 (a near wild-type P. fluorescens strain
having mutations .DELTA.pyrF and lsc::lacI.sup.QI), or its
derivatives, recovered in SOC-Soy hydrolysate medium, and plated on
selective medium (M9 glucose agar lacking uracil, Sambrook et al.,
supra). Details of the microbiological manipulations are available
in Squires et al., (2004), US Patent Application 20060008877, US
Patent Application 20080193974, and US Patent Application
20080058262, incorporated herein by reference. Colonies were first
screened by PCR and positive clones were then analyzed by
restriction digestion of miniprep plasmid DNA. Plasmid DNA of
selected clones containing inserts was sequenced, either by using
Big Dye.RTM. Terminator version 3.1 as recommended by the suppler
(Applied Biosystems/Invitrogen), or by contract with a commercial
sequencing vendor (MWG Biotech, Huntsville, Ala.). Sequence data
were assembled and analyzed using the Sequencher.TM. software (Gene
Codes Corp., Ann Arbor, Mich.).
[0137] Growth and Expression Analysis in Shake Flasks.
[0138] Production of DIG-3 toxin for characterization and insect
bioassay was accomplished by shake-flask-grown P. fluorescens
strains harboring expression constructs (e.g. clone DP2826). Seed
cultures grown in M9 medium supplemented with 1% glucose and trace
elements were used to inoculate 50 mL of defined minimal medium
with 5% glycerol (Teknova Catalog No. 3D7426, Hollister, Calif.).
Expression of the DIG-3 toxin gene via the Ptac promoter was
induced by addition of isopropyl-13-D-1-thiogalactopyranoside
(IPTG) after an initial incubation of 24 hours at 30.degree. with
shaking. Cultures were sampled at the time of induction and at
various times post-induction. Cell density was measured by optical
density at 600 nm (OD.sub.600). Other culture media suitable for
growth of Pseudomonas fluorescens may also be utilized, for
example, as described in Huang et al. (2007) and US Patent
Application 20060008877.
[0139] Cell Fractionation and SDS-PAGE Analysis of Shake Flask
Samples.
[0140] At each sampling time, the cell density of samples was
adjusted to OD.sub.600=20 and 1 mL aliquots were centrifuged at
14000.times.g for five minutes. The cell pellets were frozen at
-80.degree.. Soluble and insoluble fractions from frozen shake
flask cell pellet samples were generated using EasyLyse.TM.
Bacterial Protein Extraction Solution (EPICENTRE.RTM.
Biotechnologies, Madison, Wis.). Each cell pellet was resuspended
in 1 mL EasyLyse.TM. solution and further diluted 1:4 in lysis
buffer and incubated with shaking at room temperature for 30
minutes. The lysate was centrifuged at 14,000 rpm for 20 minutes at
4.degree. and the supernatant was recovered as the soluble
fraction. The pellet (insoluble fraction) was then resuspended in
an equal volume of phosphate buffered saline (PBS; 11.9 mM
Na.sub.2HPO.sub.4, 137 mM NaCl, 2.7 mM KCl, pH7.4).
[0141] Samples were mixed 1:1 with 2.times. Laemmli sample buffer
containing .beta.-mercaptoethanol (Sambrook et al., supra.) and
boiled for 5 minutes prior to loading onto Criterion XT.RTM.
Bis-Tris 12% gels (Bio-Rad Inc., Hercules, Calif.) Electrophoresis
was performed in the recommended XT MOPS buffer. Gels were stained
with Bio-Safe Coomassie Stain according to the manufacturer's
(Bio-Rad) protocol and imaged using the Alpha Innotech Imaging
system (San Leandro, Calif.).
[0142] Inclusion body preparation.
[0143] Cry protein inclusion body (IB) preparations were performed
on cells from P. fluorescens fermentations that produced insoluble
B.t. insecticidal protein, as demonstrated by SDS-PAGE and MALDI-MS
(Matrix Assisted Laser Desorption/Ionization Mass Spectrometry). P.
fluorescens fermentation pellets were thawed in a 37.degree. water
bath. The cells were resuspended to 25% w/v in lysis buffer (50 mM
Tris, pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt
(Ethylenediaminetetraacetic acid), 1% Triton X-100, and 5 mM
Dithiothreitol (DTT); 5 mL/L of bacterial protease inhibitor
cocktail (P8465 Sigma-Aldrich, St. Louis, Mo.) were added just
prior to use). The cells were suspended using a hand-held
homogenizer at lowest setting (Tissue Tearor, BioSpec Products,
Inc., Bartlesville, Okla.). Lysozyme (25 mg of Sigma L7651, from
chicken egg white) was added to the cell suspension by mixing with
a metal spatula, and the suspension was incubated at room
temperature for one hour. The suspension was cooled on ice for 15
minutes, then sonicated using a Branson Sonifier 250 (two 1-minute
sessions, at 50% duty cycle, 30% output). Cell lysis was checked by
microscopy. An additional 25 mg of lysozyme were added if
necessary, and the incubation and sonication were repeated. When
cell lysis was confirmed via microscopy, the lysate was centrifuged
at 11,500.times.g for 25 minutes (4.degree.) to form the IB pellet,
and the supernatant was discarded. The IB pellet was resuspended
with 100 mL lysis buffer, homogenized with the hand-held mixer and
centrifuged as above. The IB pellet was repeatedly washed by
resuspension (in 50 mL lysis buffer), homogenization, sonication,
and centrifugation until the supernatant became colorless and the
1B pellet became firm and off-white in color. For the final wash,
the B3 pellet was resuspended in sterile-filtered (0.22 .mu.m)
distilled water containing 2 mM EDTA, and centrifuged. The final
pellet was resuspended in sterile-filtered distilled water
containing 2 mM EDTA, and stored in 1 mL aliquots at
-80.degree..
[0144] SDS-PAGE analysis and quantitation of protein in 1B
preparations was done by thawing a 1 mL aliquot of IB pellet and
diluting 1:20 with sterile-filtered distilled water. The diluted
sample was then boiled with 4.times. reducing sample buffer [250 mM
Tris, pH6.8, 40% glycerol (v/v), 0.4% Bromophenol Blue (w/v), 8%
SDS (w/v) and 8%[3-Mercapto-ethanol (v/v)] and loaded onto a
Novex.RTM. 4-20% Tris-Glycine, 12+2 well gel (Invitrogen) run with
1.times. Tris/Glycine/SDS buffer (BioRad). The gel was run for 60
min at 200 volts then stained with Coomassie Blue (50% G-250/50%
R-250 in 45% methanol, 10% acetic acid), and destained with 7%
acetic acid, 5% methanol in distilled water. Quantification of
target bands was done by comparing densitometric values for the
bands against Bovine Serum Albumin (BSA) samples run on the same
gel to generate a standard curve.
[0145] Solubilization of Inclusion Bodies.
[0146] Six mL of inclusion body suspension from Pf clone DP2826
(containing 32 mg/mL of DIG-3 protein) were centrifuged on the
highest setting of an Eppendorf model 5415C microfuge
(approximately 14,000.times.g) to pellet the inclusions. The
storage buffer supernatant was removed and replaced with 25 mL of
100 mM sodium carbonate buffer, pH11, in a 50 mL conical tube.
Inclusions were resuspended using a pipette and vortexed to mix
thoroughly. The tube was placed on a gently rocking platform at
4.degree. overnight to extract the target protein. The extract was
centrifuged at 30,000.times.g for 30 min at 4.degree., and the
resulting supernatant was concentrated 5-fold using an Amicon
Ultra-15 regenerated cellulose centrifugal filter device (30,000
Molecular Weight Cutoff; Millipore). The sample buffer was then
changed to 10 mM CAPS [3-(cyclohexamino)1-propanesulfonic acid] pH
10, using disposable PD-10 columns (GE Healthcare, Piscataway,
N.J.).
[0147] Gel electrophoresis. The concentrated extract was prepared
for electrophoresis by diluting 1:50 in NuPAGE.RTM. LDS sample
buffer (Invitrogen) containing 5 mM dithiothreitol as a reducing
agent and heated at 95.degree. for 4 minutes. The sample was loaded
in duplicate lanes of a 4-12% NuPAGE.RTM. gel alongside five BSA
standards ranging from 0.2 to 2 .mu.g/lane (for standard curve
generation). Voltage was applied at 200V using MOPS SDS running
buffer (Invitrogen) until the tracking dye reached the bottom of
the gel. The gel was stained with 0.2% Coomassie Blue G-250 in 45%
methanol, 10% acetic acid, and destained, first briefly with 45%
methanol, 10% acetic acid, and then at length with 7% acetic acid,
5% methanol until the background cleared. Following destaining, the
gel was scanned with a Biorad Fluor-S MultiImager. The instrument's
Quantity One v.4.5.2 Software was used to obtain
background-subtracted volumes of the stained protein bands and to
generate the BSA standard curve that was used to calculate the
concentration of DIG-3 protein in the stock solution.
Example 5
Insecticidal Activity of Modified DIG-3 Protein Produced in
Pseudomonas fluorescens
[0148] DIG-3 B.t. insecticidal toxin was demonstrated to be active
on Lepidopteran species including the European corn borer (ECB;
Ostrinia nubilalis (Hubner)), cry1F-resistant ECB (rECB),
diamondback moth (DBM; Plutella xylostella (Linnaeus)),
cry1A-resistant DBM (rDBM), corn earworm (CEW; Helicoverpa zea
(Boddie)), black cutworm (BCW; Agrotis Ipsilon (Hufnagel)), tobacco
budworm (TBW; Heliothis virescens (Fabricius)), and cabbage looper
(CL; Trichoplusia ni (Hubner)). DIG-3 protein was also tested for
activity on fall armyworm (FAW, Spodoptera frugiperda),
Cry1F-resistant FAW (rFAW) and western corn rootworm (WCR,
Diabrotica virgifera virgifera LeConte).
[0149] Sample Preparation and Bioassays.
[0150] Inclusion body preparations in 10 mM CAPS pH10 were diluted
appropriately in 10 mM CAPS pH 10, and all bioassays contained a
control treatment consisting of this buffer, which served as a
background check for mortality or growth inhibition.
[0151] Protein concentrations in bioassay buffer were estimated by
gel electrophoresis using BSA to create a standard curve for gel
densitometry, which was measured using a BioRad imaging system
(Fluor-S MultiImager with Quantity One software version 4.5.2).
Proteins in the gel matrix were stained with Coomassie Blue-based
stain and destained before reading.
[0152] Purified proteins were tested for insecticidal activity in
bioassays conducted with neonate Lepidopteran larvae on artificial
insect diet. Larvae of BCW, CEW, CL, DBM, rDBM, ECB, FAW and TBW
were hatched from eggs obtained from a colony maintained by a
commercial insectary (Benzon Research Inc., Carlisle, Pa.). WCR
eggs were obtained from Crop Characteristics, Inc. (Farmington,
Minn.). Larvae of rECB and rFAW were hatched from eggs harvested
from proprietary colonies (Dow AgroSciences LLC, Indianapolis,
Ind.).
[0153] The bioassays were conducted in 128-well plastic trays
specifically designed for insect bioassays (C-D International,
Pitman, N.J.). Each well contained 1.0 mL of Multi-species
Lepidoptera diet (Southland Products, Lake Village, Ark.). A 40
.mu.L aliquot of protein sample was delivered by pipette onto the
1.5 cm.sup.2 diet surface of each well (26.7 .mu.L/cm.sup.2). Diet
concentrations were calculated as the amount (ng) of DIG-3 protein
per square centimeter (cm.sup.2) of surface area in the well. The
treated trays were held in a fume hood until the liquid on the diet
surface had evaporated or was absorbed into the diet.
[0154] Within a few hours of eclosion, individual larvae were
picked up with a moistened camel hair brush and deposited on the
treated diet, one larva per well. The infested wells were then
sealed with adhesive sheets of clear plastic, vented to allow gas
exchange (C-D International, Pitman, N.J.). Bioassay trays were
held under controlled environmental conditions (28.degree. C.,
.about.40% Relative Humidity, 16:8 [Light:Dark]) for 5 days, after
which the total number of insects exposed to each protein sample,
the number of dead insects, and the weight of surviving insects
were recorded. Percent mortality and percent growth inhibition were
calculated for each treatment. Growth inhibition (GI) was
calculated as follows:
GI=[1-(TWIT/TNIT)/(TWIBC/TNIBC)]
[0155] where TWIT is the Total Weight of Insects in the
Treatment,
[0156] TNIT is the Total Number of Insects in the Treatment
[0157] TWIBC is the Total Weight of Insects in the Background Check
(Buffer control), and
[0158] TNIBC is the Total Number of Insects in the Background Check
(Buffer control).
[0159] The GI.sub.50 was determined to be the concentration of
DIG-3 protein in the diet at which the GI value was 50%. The
LC.sub.50 (50% Lethal Concentration) was recorded as the
concentration of DIG-3 protein in the diet at which 50% of test
insects were killed. Statistical analysis (One-way ANOVA) was done
using IMP software (SAS, Cary, N.C.)
[0160] Table 6 presents the results of bioassay tests of DIG-3
protein on European corn borer and cry1F-resistant European corn
borer (rECB). An unexpected and surprising finding is that the rECB
test insects were as susceptible to the action of DIG-3 protein as
were the wild type ECB insects.
TABLE-US-00004 TABLE 6 LC.sub.50 and GI.sub.50 values calculated
for ECB and rECB, with Confidence Intervals (CI) of 95% Insect
LC.sub.50 (ng/cm.sup.2) 95% CI GI.sub.50 (ng/cm.sup.2) 95% CI ECB
591.9 308.1-1315.3 122.6 45.6-328.4 rECB 953.6 534.1-1953.6 270.9
53.0-1382.2
[0161] Table 7 presents the results of bioassays on a broad
spectrum of Lepidopteran and a Coleopteran pest (WCR). The DIG-3
protein has unexpected and surprising activity on diamondback moth
as well as rDBM. Further the DIG-3 Cry protein is effective in
controlling the growth of several other Lepidopteran insects.
TABLE-US-00005 TABLE 7 Insecticidal and growth inhibitory effects
of DIG-3 protein ingested by test insects Test Response at Insect
9000 ng/cm.sup.2 Statistical Analysis* DBM 100% mortality rDBM 100%
mortality CL 75% mortality, significant GI (GI) P < 0.001, df =
1, .alpha. = 0.05 CEW Significant GI (GI) P = 0.02, df = 1, .alpha.
= 0.05 TBW Visible GI, some mortality Not available BCW Visible GI
Not available FAW No activity observed rFAW No activity observed
WCR No activity observed *GI = Growth Inhibition. P-value = test
statistic. df = Degrees of Freedom, .alpha. (alpha) level 0.05 =
the level of test significance.
Example 6
Agrobacterium Transformation
[0162] Standard cloning methods are used in the construction of
binary plant transformation and expression plasmids. Restriction
endonucleases and T4 DNA Ligase are obtained from NEB. Plasmid
preparations are performed using the NucleoSpin.RTM. Plasmid
Preparation kit or the NucleoBond.RTM. AX Xtra Midi kit (both from
Macherey-Nagel), following the instructions of the manufacturers.
DNA fragments are purified using the QIAquick.RTM. PCR Purification
Kit or the QIAEX II.RTM. Gel Extraction Kit (both from Qiagen)
after gel isolation.
[0163] DNA fragments comprising nucleotide sequences that encode
the DIG-3 protein in native or modified forms, or fragments
thereof, may be synthesized by a commercial vendor (e.g. DNA2.0,
Menlo Park, Calif.) and supplied as cloned fragments in standard
plasmid vectors, or may be obtained by standard molecular biology
manipulation of other constructs containing appropriate nucleotide
sequences. Unique restriction sites internal to a DIG-3 coding
region may be identified and DNA fragments comprising the sequences
between the restriction sites of the DIG-3 coding region may be
synthesized, each such fragment encoding a specific deletion,
insertion or other DIG-3 variation. The DNA fragments encoding the
modified DIG-3 fragments may be joined to other DIG-3 coding region
fragments or other Cry coding region fragments at appropriate
restriction sites to obtain a coding region encoding the desired
full-length DIG-3 protein, deleted or variant DIG-3 protein, or
fused protein. For example, one may identify an appropriate
restriction recognition site at the start of a first DIG-3 coding
region, and a second restriction site internal to the DIG-3 coding
region. Cleavage of this first DIG-3 coding region at these
restriction sites would generate a DNA fragment comprising part of
the first DIG-3 coding region. A second DNA fragment flanked by
analogously-situated compatible restriction sites specific for
another DIG-3 coding region or other Cry coding region may be used
in combination with the first DNA restriction fragment to construct
a variant or fused clone.
[0164] In a non-limiting example, a basic cloning strategy may be
to subclone full length or modified DIG-3 coding sequences (CDS)
into a plant expression plasmid at NcoI and SacI restriction sites.
The resulting plant expression cassettes containing the appropriate
DIG-3 coding region under the control of plant expression elements,
(e.g., plant expressible promoters, 3' terminal transcription
termination and polyadenylate addition determinants, and the like)
are subcloned into a binary vector plasmid, utilizing, for example,
Gateway.RTM. technology or standard restriction enzyme fragment
cloning procedures. LR Clonase.TM. (Invitrogen) for example, may be
used to recombine the full length and modified gene plant
expression cassettes into a binary plant transformation plasmid if
the Gateway.RTM. technology is utilized. It is convenient to employ
a binary plant transformation vector that harbors a bacterial gene
that confers resistance to the antibiotic spectinomycin when the
plasmid is present in E. coli and Agrobacterium cells. It is also
convenient to employ a binary vector plasmid that contains a
plant-expressible selectable marker gene that is functional in the
desired host plants. Examples of plant-expressible selectable
marker genes include but are not limited those that encode the
aminoglycoside phosphotransferase gene (aphII) of transposon Tn5,
which confers resistance to the antibiotics kanamycin, neomycin and
G418, as well as those genes which code for resistance or tolerance
to glyphosate; hygromycin; methotrexate; phosphinothricin
(bialaphos), imidazolinones, sulfonylureas and triazolopyrimidine
herbicides, such as chlorosulfuron, bromoxynil, dalapon and the
like.
[0165] Electro-competent cells of Agrobacterium tumefaciens strain
Z707S (a streptomycin-resistant derivative of Z707; Hepburn et al.,
1985) are prepared and transformed using electroporation (Weigel
and Glazebrook, 2002). After electroporation, 1 mL of YEP broth
(gm/L: yeast extract, 10; peptone, 10; NaCl, 5) are added to the
cuvette and the cell-YEP suspension is transferred to a 15 mL
culture tube for incubation at 28.degree. in a water bath with
constant agitation for 4 hours. The cells are plated on YEP plus
agar (25 gm/L) with spectinomycin (200 .mu.g/mL) and streptomycin
(250 .mu.g/mL) and the plates are incubated for 2-4 days at
28.degree.. Well separated single colonies are selected and
streaked onto fresh YEP+agar plates with spectinomycin and
streptomycin as before, and incubated at 28.degree. for 1-3
days.
[0166] The presence of the DIG-3 gene insert in the binary plant
transformation vector is performed by PCR analysis using
vector-specific primers with template plasmid DNA prepared from
selected Agrobacterium colonies. The cell pellet from a 4 mL
aliquot of a 15 mL overnight culture grown in YEP with
spectinomycin and streptomycin as before is extracted using Qiagen
Spin.RTM. Mini Preps, performed per manufacturer's instructions.
Plasmid DNA from the binary vector used in the Agrobacterium
electroporation transformation is included as a control. The PCR
reaction is completed using Taq DNA polymerase from Invitrogen per
manufacture's instructions at 0.5.times. concentrations. PCR
reactions are carried out in a MJ Research Peltier Thermal Cycler
programmed with the following conditions: Step 1) 94.degree. for 3
minutes; Step 2) 94.degree. for 45 seconds; Step 3) 55.degree. for
30 seconds; Step 4) 72.degree. for 1 minute per kb of expected
product length; Step 5) 29 times to Step 2; Step 6) 72.degree. for
10 minutes. The reaction is maintained at 4.degree. after cycling.
The amplification products are analyzed by agarose gel
electrophoresis (e.g. 0.7% to 1% agarose, w/v) and visualized by
ethidium bromide staining. A colony is selected whose PCR product
is identical to the plasmid control.
[0167] Alternatively, the plasmid structure of the binary plant
transformation vector containing the DIG-3 gene insert is performed
by restriction digest fingerprint mapping of plasmid DNA prepared
from candidate Agrobacterium isolates by standard molecular biology
methods well known to those skilled in the art of Agrobacterium
manipulation.
[0168] Those skilled in the art of obtaining transformed plants via
Agrobacterium-mediated transformation methods will understand that
other Agrobacterium strains besides Z707S may be used to advantage,
and the choice of strain may depend upon the identity of the host
plant species to be transformed.
Example 7
Production of DIG-3 B.t. Insecticidal Proteins and Variants in
Dicot Plants
[0169] Arabidopsis Transformation.
[0170] Arabidopsis thaliana Col-01 is transformed using the floral
dip method (Weigel and Glazebrook, 2002). The selected
Agrobacterium colony is used to inoculate 1 mL to 15 mL cultures of
YEP broth containing appropriate antibiotics for selection. The
culture is incubated overnight at 28.degree. with constant
agitation at 220 rpm. Each culture is used to inoculate two 500 mL
cultures of YEP broth containing appropriate antibiotics for
selection and the new cultures are incubated overnight at
28.degree. with constant agitation. The cells are pelleted at
approximately 8700.times.g for 10 minutes at room temperature, and
the resulting supernatant is discarded. The cell pellet is gently
resuspended in 500 mL of infiltration media containing: 1/2.times.
Murashige and Skoog salts (Sigma-Aldrich)/Gamborg's B5 vitamins
(Gold BioTechnology, St. Louis, Mo.), 10% (w/v) sucrose, 0.044
.mu.M benzylaminopurine (10 .mu.L/liter of 1 mg/mL stock in DMSO)
and 300 .mu.L/liter Silwet L-77. Plants approximately 1 month old
are dipped into the media for 15 seconds, with care taken to assure
submergence of the newest inflorescence. The plants are then laid
on their sides and covered (transparent or opaque) for 24 hours,
washed with water, and placed upright. The plants are grown at
22.degree., with a 16-hour light/8-hour dark photoperiod.
Approximately 4 weeks after dipping, the seeds are harvested.
[0171] Arabidopsis Growth and Selection.
[0172] Freshly harvested T1 seed is allowed to dry for at least 7
days at room temperature in the presence of desiccant. Seed is
suspended in a 0.1% agar/water (Sigma-Aldrich) solution and then
stratified at 4.degree. for 2 days. To prepare for planting,
Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.) in
10.5 inch.times.21 inch germination trays (T.O. Plastics Inc.,
Clearwater, Minn.) is covered with fine vermiculite, sub-irrigated
with Hoagland's solution (Hoagland and Amon, 1950) until wet, then
allowed to drain for 24 hours. Stratified seed is sown onto the
vermiculite and covered with humidity domes (KORD Products,
Bramalea, Ontario, Canada) for 7 days. Seeds are germinated and
plants are grown in a Conviron (Models CMP4030 or CMP3244;
Controlled Environments Limited, Winnipeg, Manitoba, Canada) under
long day conditions (16 hours light/8 hours dark) at a light
intensity of 120-150 .mu.mol/m.sup.2 sec under constant temperature
(22.degree.) and humidity (40-50%). Plants are initially watered
with Hoagland's solution and subsequently with deionized water to
keep the soil moist but not wet.
[0173] The domes are removed 5-6 days post sowing and plants are
sprayed with a chemical selection agent to kill plants germinated
from nontransformed seeds. For example, if the plant expressible
selectable marker gene provided by the binary plant transformation
vector is a pat or bar gene (Wehrmann et al., 1996), transformed
plants may be selected by spraying with a 1000.times. solution of
Finale (5.78% glufosinate ammonium, Farnam Companies Inc., Phoenix,
Ariz.). Two subsequent sprays are performed at 5-7 day intervals.
Survivors (plants actively growing) are identified 7-10 days after
the final spraying and transplanted into pots prepared with
Sunshine Mix LP5. Transplanted plants are covered with a humidity
dome for 3-4 days and placed in a Conviron under the
above-mentioned growth conditions.
[0174] Those skilled in the art of dicot plant transformation will
understand that other methods of selection of transformed plants
are available when other plant expressible selectable marker genes
(e.g. herbicide tolerance genes) are used.
[0175] Insect Bioassays of Transgenic Arabidopsis.
[0176] Transgenic Arabidopsis lines expressing modified Cry
proteins are demonstrated to be active against sensitive insect
species in artificial diet overlay assays. Protein extracted from
transgenic and non-transgenic Arabidopsis lines is quantified by
appropriate methods and sample volumes are adjusted to normalize
protein concentration. Bioassays are conducted on artificial diet
as described above. Non-transgenic Arabidopsis and/or buffer and
water are included in assays as background check treatments.
Example 8
Agrobacterium Transformation for Eneration of Superbinary
Vectors
[0177] The Agrobacterium superbinary system is conveniently used
for transformation of monocot plant hosts. Methodologies for
constructing and validating superbinary vectors are well disclosed
and incorporated herein by reference (Operating Manual for Plasmid
pSB 1, Version 3.1, available from Japan Tobacco, Inc., Tokyo,
Japan). Standard molecular biological and microbiological methods
are used to generate superbinary plasmids. Verification/validation
of the structure of the superbinary plasmid is done using
methodologies as described above for binary vectors, and may be
modified as suggested in the Operating Manual for Plasmid pSB
1.
Example 9
Production of DIG-3 B.t. Insecticidal Proteins and Variants in
Monocot Plants
[0178] Agrobacterium-Mediated Transformation of Maize. Seeds from a
High II F.sub.1 cross (Armstrong et al., 1991) are planted into
5-gallon-pots containing a mixture of 95% Metro-Mix 360 soilless
growing medium (Sun Gro Horticulture, Bellevue, Wash.) and 5%
clay/loam soil. The plants are grown in a greenhouse using a
combination of high pressure sodium and metal halide lamps with a
16:8 hour Light:Dark photoperiod. For obtaining immature F.sub.2
embryos for transformation, controlled sib-pollinations are
performed. Immature embryos are isolated at 8-10 days
post-pollination when embryos are approximately 1.0 to 2.0 mm in
size.
[0179] Infection and Co-Cultivation.
[0180] Maize ears are surface sterilized by scrubbing with liquid
soap, immersing in 70% ethanol for 2 minutes, and then immersing in
20% commercial bleach (0.1% sodium hypochlorite) for 30 minutes
before being rinsed with sterile water. A suspension of
Agrobacterium cells containing a superbinary vector is prepared by
transferring 1-2 loops of bacteria grown on YEP solid medium
containing 100 mg/L spectinomycin, 10 mg/L tetracycline, and 250
mg/L streptomycin at 28.degree. for 2-3 days into 5 mL of liquid
infection medium (LS Basal Medium (Linsmaier and Skoog, 1965), N6
vitamins (Chu et al., 1975), 1.5 mg/L 2,4-Dichlorophenoxyacetic
acid (2,4-D), 68.5 gm/L sucrose, 36.0 gm/L glucose, 6 mM L-proline,
pH 5.2) containing 100 .mu.M acetosyringone. The solution was
vortexed until a uniform suspension was achieved, and the
concentration is adjusted to a final density of about 200 Klett
units, using a Klett-Summerson colorimeter with a purple filter, or
an optical density of approximately 0.4 at 550 nm. Immature embryos
are isolated directly into a micro centrifuge tube containing 2 mL
of the infection medium. The medium is removed and replaced with 1
mL of the Agrobacterium solution with a density of 200 Klett units,
and the Agrobacterium and embryo solution is incubated for 5
minutes at room temperature and then transferred to co-cultivation
medium (LS Basal Medium, N6 vitamins, 1.5 mg/L 2,4-D, 30.0 gm/L
sucrose, 6 mM L-proline, 0.85 mg/L AgNO.sub.3, 100 .mu.M
acetosyringone, 3.0 gm/L Gellan gum (PhytoTechnology Laboratories.,
Lenexa, Kans.), pH 5.8) for 5 days at 25.degree. under dark
conditions.
[0181] After co-cultivation, the embryos are transferred to
selective medium after which transformed isolates are obtained over
the course of approximately 8 weeks. For selection of maize tissues
transformed with a superbinary plasmid containing a plant
expressible pat or bar selectable marker gene, an LS based medium
(LS Basal medium, N6 vitamins, 1.5 mg/L 2,4-D, 0.5 gm/L MES
(2-(N-morpholino)ethanesulfonic acid monohydrate; PhytoTechnologies
Labr.), 30.0 gm/L sucrose, 6 mM L-proline, 1.0 mg/L AgNO.sub.3, 250
mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) is used with
Bialaphos (Gold BioTechnology). The embryos are transferred to
selection media containing 3 mg/L Bialaphos until embryogenic
isolates were obtained. Recovered isolates are bulked up by
transferring to fresh selection medium at 2-week intervals for
regeneration and further analysis.
[0182] Those skilled in the art of maize transformation will
understand that other methods of selection of transformed plants
are available when other plant expressible selectable marker genes
(e.g. herbicide tolerance genes) are used.
[0183] Regeneration and Seed Production.
[0184] For regeneration, the cultures are transferred to "28"
induction medium (MS salts and vitamins, 30 gm/L sucrose, 5 mg/L
Benzylaminopurine, 0.25 mg/L 2,4-D, 3 mg/L Bialaphos, 250 mg/L
cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) for 1 week under low-light
conditions (14 .mu.m.sup.-2s.sup.-1) then 1 week under high-light
conditions (approximately 89 .mu.Em.sup.-2 s.sup.-1). Tissues are
subsequently transferred to "36" regeneration medium (same as
induction medium except lacking plant growth regulators). When
plantlets grow to 3-5 cm in length, they were transferred to glass
culture tubes containing SHGA medium (Schenk and Hildebrandt salts
and vitamins (1972); PhytoTechnologies Labr.), 1.0 gm/L
myo-inositol, 10 gm/L sucrose and 2.0 gm/L Gellan gum, pH 5.8) to
allow for further growth and development of the shoot and roots.
Plants are transplanted to the same soil mixture as described
earlier herein and grown to flowering in the greenhouse. Controlled
pollinations for seed production are conducted.
Example 10
Bioassay of Transgenic Maize
[0185] Bioactivity of the DIG-3 protein and variants produced in
plant cells is demonstrated by conventional bioassay methods (see,
for example Huang et al., 2006). One is able to demonstrate
efficacy, for example, by feeding various plant tissues or tissue
pieces derived from a plant producing a DIG-3 toxin to target
insects in a controlled feeding environment. Alternatively, protein
extracts may be prepared from various plant tissues derived from a
plant producing the DIG-3 toxin and incorporate the extracted
proteins in an artificial diet bioassay as previously described
herein. It is to be understood that the results of such feeding
assays are to be compared to similarly conducted bioassays that
employ appropriate control tissues from host plants that do not
produce the DIG-3 protein or variants, or to other control
samples.
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Sequence CWU 1
1
713771DNABacillus thuringiensismisc_feature(1)..(3)translation
start codon specifying Met 1atgacttcaa ataggaaaaa tgagaatgaa
attataaatg ctttatcgat tccaacggta 60tcgaatcctt ccacgcaaat gaatctatca
ccagatgctc gtattgaaga tagcttgtgt 120gtagccgagg tgaacaatat
tgatccattt gttagcgcat caacagtcca aacgggtata 180aacatagctg
gtagaatatt gggcgtatta ggtgtgccgt ttgctggaca actagctagt
240ttttatagtt ttcttgttgg tgaattatgg cctagtggca gagatccatg
ggaaattttc 300ctggaacatg tagaacaact tataagacaa caagtaacag
aaaatactag gaatacggct 360attgctcgat tagaaggtct aggaagaggc
tatagatctt accagcaggc tcttgaaact 420tggttagata accgaaatga
tgcaagatca agaagcatta ttcttgagcg ctatgttgct 480ttagaacttg
acattactac tgctataccg cttttcagaa tacgaaatca agaggttcca
540ttattaatgg tatatgctca agctgcaaat ttacacctat tattattgag
ggacgcatcc 600ctttttggta gtgaatgggg gacggcatct tccgatgtta
accaatatta ccaagaacaa 660atcagatata cagaggaata ttctaaccat
tgcgtacaat ggtataatac ggggctaaat 720aacttaagag ggacaaatgc
tgaaagttgg gtacggtata atcaattccg cagagaccta 780acattagggg
tattagatct agtggcccta ttcccaagtt atgacactcg cacttatcca
840atcaatacga gtgctcagtt aacaagagaa gtttatacag acgcaattgg
gaccgtacat 900ccgagtcaag cttttgcaag tacgacttgg tttaataata
atgcaccatc gttttctgcc 960atagaagctg ccgttatcag gcctccgcat
ctacttgatt ttccagaaca acttacaatt 1020tacagcacat taagtcgatg
gagtaacact cagtttatga atatatgggc aggtcataga 1080cttgaatccc
gcccaatagc agggtcatta aatacctcta cacaaggatc taccaatact
1140tctattaatc ctgtaacatt acagtttacg tctcgagaca tttataggac
tgaatcattg 1200gcagggctaa atatatttat aactcaacct gttaatgggg
ttccttgggt tagatttaat 1260tggagaaatc ccctgaattc tcttagaggt
agccttctct atacgatagg gtatactgga 1320gttgggacgc aattacaaga
ttcagaaact gaattacccc cagaaacaac agaacgacca 1380aattatgaat
catatagtca tagattatct catataggac tcatttcatc atctcatgtg
1440agagcattgg tatattcttg gacgcaccgt agtgcagatc gtacgaatac
gattggacca 1500aatagaatta ctcaaattcc tgcagtgaag ggaagatttc
tttttaatgg ctctgtaatt 1560tcaggaccag gatttactgg tggagacgta
gttagattga ataggaataa tggtaatatt 1620caaaatagag ggtatattga
agttccaatt caattcacgt cgacatctac cagatatcga 1680gttcgagtac
gttatgcttc tgtaacctcg attgagctca atgttaattg gggcaattca
1740tcaattttta cgaacacatt accagcaaca gctgcatcat tagataatct
acaatcaggg 1800gattttggtt atgttgaaat caacaatgct tttacatccg
caacaggtaa tatagtaggt 1860gttagaaatt ttagtgcaaa tgcagaggta
ataatagaca gatttgaatt tatcccagtt 1920actgcaacct tcgaggcaaa
atatgattta gaaagagcac aaaaggcggt gaatgctctg 1980tttacttcta
caaatccaag aagattgaag acagatgtga cagattatca tattgaccaa
2040gtgtccaatc tggtggtatg tttatcagat gaattttgct tggatgagaa
gcgagaatta 2100tttgagaaag tgaaatatgc gaagcgactc agtgatgaaa
gaaacttact ccaagatcca 2160aacttcacat tcatcaatgg gcaaccaagt
tttgcatcca tcgatggaca atcaaacttc 2220acctctatta atgagctatc
taatcatgga tggtggggca gtgcgaatgt taccattcag 2280gaagggaatg
acgtatttaa agagaattac gtcacactac cgggtacttt taatgagtgt
2340tatccaaatt atttatatca aaaaatagga gagtcagaat taaaggctta
tacgcgctat 2400caattaagag ggtatattga agatagtcaa gatctagaga
tttatttaat tcgttacaat 2460gcaaagcatg aaacattaaa tgttccaggt
accgagtccc tatggccgct ttcagttgaa 2520agcccaatcg gaaggtgcgg
agaaccaaat cgatgcgcac cacattttgg atggaatcct 2580gatctagatt
gttcctgcag agatagagaa aaatgtgcgc atcattccca tcatttcact
2640ttggatattg atgttggatg cacagacttg caagaggatc taggcgtgtg
ggttgtattc 2700aagattaaga cgcaggaagg ttatgcaaga ttaggaaatc
tggaatttat cgaagagaaa 2760ccattaattg gagaagcact gtctcgtgtg
aagagagcgg aaaaaaaatg gagagacaaa 2820agggaaaaac tacaagtgga
aacaaaacga gtatatatag acgcaaaaga agctgtggat 2880gctttattcg
tagattctca atatgataga ttacaagcag atacaaacat cggtatgatt
2940catgcggcag atagacttgt tcatcggatc cacgaggctt atcttccaga
actacctttc 3000attccaggaa taaatgtggt gatttttgaa gaattagaaa
accgtatttc tactgcattt 3060tccttatatg atgcgagaaa tgtcattaaa
aatggcgatt tcaataatgg attgacatgc 3120tggaacgtga aagggcatgt
agaggtacag cagctgaaca atcatcgttc ggtccttgtc 3180atcccggaat
gggaagcaga agtttcacaa aaggtgcgcg tctgtccagg tcgtggctat
3240attcttcgtg tcacagcgta caaagaggga tatggggaag gctgcgtaac
tattcatgaa 3300gtcgataata atacagacca attgaagttt agcaactgtg
agaaaggaca agtatatcca 3360ggtaatacga tagcatgtaa tgattataat
aagaatcatg gtgcgaatgc atgtagttct 3420cgtaatcgtg gatatgacga
attctatgga aacaccccag ctgattattc tgcaaatcaa 3480aaagaatacg
ggggtgcgta cacttcccac aatcatgcat atggcgaatc ttatgaaagt
3540aattcgtcca taccagctga ttatgcgccg gtttatgaag aagaagcgta
tacacatgga 3600cgaagaggta attcttgtga atataacaga gggtatacac
cattaccagc tggttatgtg 3660acagcagagt tagaatactt cccagaaacg
gatacagtat gggttgagat tggagaaacg 3720gaaggaacat ttatcgtgga
caatgtggaa ttactcctta tggaggaata g 377121256PRTBacillus
thuringiensis 2Met Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn
Ala Leu Ser 1 5 10 15 Ile Pro Thr Val Ser Asn Pro Ser Thr Gln Met
Asn Leu Ser Pro Asp 20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Val
Ala Glu Val Asn Asn Ile Asp 35 40 45 Pro Phe Val Ser Ala Ser Thr
Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val
Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser 65 70 75 80 Phe Tyr Ser
Phe Leu Val Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro 85 90 95 Trp
Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile Arg Gln Gln Val 100 105
110 Thr Glu Asn Thr Arg Asn Thr Ala Ile Ala Arg Leu Glu Gly Leu Gly
115 120 125 Arg Gly Tyr Arg Ser Tyr Gln Gln Ala Leu Glu Thr Trp Leu
Asp Asn 130 135 140 Arg Asn Asp Ala Arg Ser Arg Ser Ile Ile Leu Glu
Arg Tyr Val Ala 145 150 155 160 Leu Glu Leu Asp Ile Thr Thr Ala Ile
Pro Leu Phe Arg Ile Arg Asn 165 170 175 Gln Glu Val Pro Leu Leu Met
Val Tyr Ala Gln Ala Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg
Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly Thr 195 200 205 Ala Ser Ser
Asp Val Asn Gln Tyr Tyr Gln Glu Gln Ile Arg Tyr Thr 210 215 220 Glu
Glu Tyr Ser Asn His Cys Val Gln Trp Tyr Asn Thr Gly Leu Asn 225 230
235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg Tyr Asn Gln
Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala
Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr
Ser Ala Gln Leu Thr 275 280 285 Arg Glu Val Tyr Thr Asp Ala Ile Gly
Thr Val His Pro Ser Gln Ala 290 295 300 Phe Ala Ser Thr Thr Trp Phe
Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala
Val Ile Arg Pro Pro His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu
Thr Ile Tyr Ser Thr Leu Ser Arg Trp Ser Asn Thr Gln Phe 340 345 350
Met Asn Ile Trp Ala Gly His Arg Leu Glu Ser Arg Pro Ile Ala Gly 355
360 365 Ser Leu Asn Thr Ser Thr Gln Gly Ser Thr Asn Thr Ser Ile Asn
Pro 370 375 380 Val Thr Leu Gln Phe Thr Ser Arg Asp Ile Tyr Arg Thr
Glu Ser Leu 385 390 395 400 Ala Gly Leu Asn Ile Phe Ile Thr Gln Pro
Val Asn Gly Val Pro Trp 405 410 415 Val Arg Phe Asn Trp Arg Asn Pro
Leu Asn Ser Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr
Thr Gly Val Gly Thr Gln Leu Gln Asp Ser 435 440 445 Glu Thr Glu Leu
Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser
His Arg Leu Ser His Ile Gly Leu Ile Ser Ser Ser His Val 465 470 475
480 Arg Ala Leu Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn
485 490 495 Thr Ile Gly Pro Asn Arg Ile Thr Gln Ile Pro Ala Val Lys
Gly Arg 500 505 510 Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly
Phe Thr Gly Gly 515 520 525 Asp Val Val Arg Leu Asn Arg Asn Asn Gly
Asn Ile Gln Asn Arg Gly 530 535 540 Tyr Ile Glu Val Pro Ile Gln Phe
Thr Ser Thr Ser Thr Arg Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr
Ala Ser Val Thr Ser Ile Glu Leu Asn Val Asn 565 570 575 Trp Gly Asn
Ser Ser Ile Phe Thr Asn Thr Leu Pro Ala Thr Ala Ala 580 585 590 Ser
Leu Asp Asn Leu Gln Ser Gly Asp Phe Gly Tyr Val Glu Ile Asn 595 600
605 Asn Ala Phe Thr Ser Ala Thr Gly Asn Ile Val Gly Val Arg Asn Phe
610 615 620 Ser Ala Asn Ala Glu Val Ile Ile Asp Arg Phe Glu Phe Ile
Pro Val 625 630 635 640 Thr Ala Thr Phe Glu Ala Lys Tyr Asp Leu Glu
Arg Ala Gln Lys Ala 645 650 655 Val Asn Ala Leu Phe Thr Ser Thr Asn
Pro Arg Arg Leu Lys Thr Asp 660 665 670 Val Thr Asp Tyr His Ile Asp
Gln Val Ser Asn Leu Val Val Cys Leu 675 680 685 Ser Asp Glu Phe Cys
Leu Asp Glu Lys Arg Glu Leu Phe Glu Lys Val 690 695 700 Lys Tyr Ala
Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro 705 710 715 720
Asn Phe Thr Phe Ile Asn Gly Gln Pro Ser Phe Ala Ser Ile Asp Gly 725
730 735 Gln Ser Asn Phe Thr Ser Ile Asn Glu Leu Ser Asn His Gly Trp
Trp 740 745 750 Gly Ser Ala Asn Val Thr Ile Gln Glu Gly Asn Asp Val
Phe Lys Glu 755 760 765 Asn Tyr Val Thr Leu Pro Gly Thr Phe Asn Glu
Cys Tyr Pro Asn Tyr 770 775 780 Leu Tyr Gln Lys Ile Gly Glu Ser Glu
Leu Lys Ala Tyr Thr Arg Tyr 785 790 795 800 Gln Leu Arg Gly Tyr Ile
Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu 805 810 815 Ile Arg Tyr Asn
Ala Lys His Glu Thr Leu Asn Val Pro Gly Thr Glu 820 825 830 Ser Leu
Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 835 840 845
Pro Asn Arg Cys Ala Pro His Phe Gly Trp Asn Pro Asp Leu Asp Cys 850
855 860 Ser Cys Arg Asp Arg Glu Lys Cys Ala His His Ser His His Phe
Thr 865 870 875 880 Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Gln Glu
Asp Leu Gly Val 885 890 895 Trp Val Val Phe Lys Ile Lys Thr Gln Glu
Gly Tyr Ala Arg Leu Gly 900 905 910 Asn Leu Glu Phe Ile Glu Glu Lys
Pro Leu Ile Gly Glu Ala Leu Ser 915 920 925 Arg Val Lys Arg Ala Glu
Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu 930 935 940 Gln Val Glu Thr
Lys Arg Val Tyr Ile Asp Ala Lys Glu Ala Val Asp 945 950 955 960 Ala
Leu Phe Val Asp Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn 965 970
975 Ile Gly Met Ile His Ala Ala Asp Arg Leu Val His Arg Ile His Glu
980 985 990 Ala Tyr Leu Pro Glu Leu Pro Phe Ile Pro Gly Ile Asn Val
Val Ile 995 1000 1005 Phe Glu Glu Leu Glu Asn Arg Ile Ser Thr Ala
Phe Ser Leu Tyr 1010 1015 1020 Asp Ala Arg Asn Val Ile Lys Asn Gly
Asp Phe Asn Asn Gly Leu 1025 1030 1035 Thr Cys Trp Asn Val Lys Gly
His Val Glu Val Gln Gln Leu Asn 1040 1045 1050 Asn His Arg Ser Val
Leu Val Ile Pro Glu Trp Glu Ala Glu Val 1055 1060 1065 Ser Gln Lys
Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg 1070 1075 1080 Val
Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile 1085 1090
1095 His Glu Val Asp Asn Asn Thr Asp Gln Leu Lys Phe Ser Asn Cys
1100 1105 1110 Glu Lys Gly Gln Val Tyr Pro Gly Asn Thr Ile Ala Cys
Asn Asp 1115 1120 1125 Tyr Asn Lys Asn His Gly Ala Asn Ala Cys Ser
Ser Arg Asn Arg 1130 1135 1140 Gly Tyr Asp Glu Phe Tyr Gly Asn Thr
Pro Ala Asp Tyr Ser Ala 1145 1150 1155 Asn Gln Lys Glu Tyr Gly Gly
Ala Tyr Thr Ser His Asn His Ala 1160 1165 1170 Tyr Gly Glu Ser Tyr
Glu Ser Asn Ser Ser Ile Pro Ala Asp Tyr 1175 1180 1185 Ala Pro Val
Tyr Glu Glu Glu Ala Tyr Thr His Gly Arg Arg Gly 1190 1195 1200 Asn
Ser Cys Glu Tyr Asn Arg Gly Tyr Thr Pro Leu Pro Ala Gly 1205 1210
1215 Tyr Val Thr Ala Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr Val
1220 1225 1230 Trp Val Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val
Asp Asn 1235 1240 1245 Val Glu Leu Leu Leu Met Glu Glu 1250 1255
33771DNAArtificial SequenceSynthetic molecule 3atgacctcca
accgtaagaa cgaaaacgaa atcatcaatg ccctcagcat acccaccgtt 60agcaatccct
ccactcagat gaacctcagc ccagatgcca gaattgagga ctccctctgc
120gtggcagagg tgaacaacat tgaccctttt gtctcagcct ccactgttca
aactggcatc 180aacattgctg ggaggattct tggtgtcctc ggtgtcccat
tcgctggcca gctcgcttct 240ttctactcct ttctggttgg tgagctgtgg
ccttccggac gtgacccttg ggaaatcttc 300cttgagcacg ttgaacagct
gattaggcag caagttactg aaaacacacg taacacagcc 360attgccagac
tggagggtct cggcagagga tatcgcagct atcagcaagc actggaaacg
420tggctggata acagaaatga tgcaaggtca cgctccatca ttctcgaacg
ctacgtggca 480ctggaacttg acataacaac agccatacct ctgttcagaa
tcagaaacca agaagtccct 540ctgttgatgg tctatgctca agcagccaac
ttgcacttgc ttcttttgag ggatgcctct 600ctgttcggga gcgaatgggg
aaccgcttcc agcgacgtga atcagtacta tcaagagcag 660ataagataca
cggaagagta ctcaaatcac tgcgttcagt ggtacaatac tggcttgaac
720aatctgcgtg gcaccaatgc cgaatcttgg gtgcgttaca atcagttcag
aagagacctc 780acacttgggg tgctcgacct cgttgctctc tttccaagct
atgacacaag gacttatccc 840atcaatactt cagcacagct gacgagggaa
gtgtacacag acgcaatagg cacagttcac 900ccctcccaag cctttgcctc
cacgacatgg ttcaacaaca atgcaccctc attctcagcc 960atagaagcag
ctgtgattcg tccaccccat ttgctggact ttccagagca gttgacgatc
1020tatagcaccc tttcaaggtg gtcaaacacg caattcatga acatttgggc
tggtcataga 1080cttgagtcaa ggccaatcgc tggttctctt aacacatcaa
cccaaggctc caccaacacc 1140tccatcaacc cagtcaccct ccagttcacc
agcagagaca tctatcgcac agaatccctt 1200gctggactca acatcttcat
tacacagcca gtcaatggag tcccgtgggt gaggttcaac 1260tggaggaatc
ccttgaactc acttagggga agccttcttt acaccatagg ttacacgggt
1320gtgggaacgc agttgcaaga ttcagagact gaactgcctc ccgagaccac
cgaacgtccc 1380aactatgaat catactccca ccgtctgtcc cacattggtc
ttatcagctc cagccacgtg 1440cgtgccctcg tctactcttg gacgcatcgc
tccgctgata ggaccaatac cataggtccg 1500aatagaatca cccagatccc
agcagtgaag ggcagattct tgttcaatgg ctctgtcatt 1560tctggtcctg
gtttcactgg tggtgacgtc gttcgcttga acagaaacaa tgggaacatt
1620caaaaccgtg gttacatcga ggtgcccatc cagtttactt ctacatcaac
acgttacaga 1680gttcgcgtca gatacgcctc tgtgacttct attgaattga
acgtgaactg gggaaacagc 1740tctatcttca ctaacacact tccagccacc
gcagcttcac tggacaatct tcagtctggg 1800gactttggtt atgtggagat
caacaatgct ttcacgtctg ccactgggaa cattgttggt 1860gtgagaaact
tctctgccaa tgccgaggtg atcatagata gatttgagtt cattccagtt
1920actgccacct ttgaggcaaa gtacgatctt gagagagcac agaaggctgt
caacgctctg 1980ttcacaagca ccaacccaag acgtctgaaa actgatgtga
cagactatca catcgatcaa 2040gttagcaact tggtggtttg cctctcagat
gagttctgct tggatgagaa gagggaactc 2100tttgagaagg ttaagtatgc
aaaacgcctt tcagacgaaa gaaacttgct gcaagacccc 2160aactttacgt
tcatcaatgg acaacctagc ttcgcttcca ttgatggtca aagcaacttt
2220acttctatca atgagttgtc taatcacggc tggtggggaa gcgcaaatgt
cacaattcaa 2280gaaggcaacg atgtgttcaa agagaactac gtgacgctgc
ctggaacatt caatgagtgt 2340tatccgaact acttgtatca gaagattggt
gaatcagagc tgaaggctta cactcgctat 2400cagctgcgtg gttacatcga
ggactcccaa gaccttgaaa tctatctcat ccgctacaac 2460gctaaacacg
agacattgaa tgttcctgga acagagtccc tttggcctct gtcagttgag
2520tctcctatag ggaggtgtgg cgaacctaac agatgtgctc ctcactttgg
gtggaatccc 2580gatttggatt gctcttgtag ggatcgcgag aagtgcgctc
accattcaca ccactttacg 2640ctggacatag atgttggctg cacggacctt
caagaggatc tcggtgtgtg ggtcgtgttc 2700aagatcaaaa ctcaagaggg
ctacgcaaga ctcgggaatc ttgagttcat tgaggagaag 2760cctctcattg
gcgaggctct ctctagggtg aagagagccg agaagaagtg gcgtgacaag
2820agggagaaac ttcaagttga
gacgaagagg gtgtacattg atgctaaaga ggctgttgat 2880gcactgtttg
tggattcaca gtatgatagg ctccaagctg acacaaacat tggaatgatt
2940catgcagcag atcgcctcgt gcatcgcatc catgaggctt atcttccgga
actcccgttc 3000atccctggga tcaatgttgt catctttgag gagcttgaga
accgcatatc cacagccttt 3060tccctctacg atgctagaaa tgttatcaag
aatggcgatt tcaacaatgg cttgacgtgt 3120tggaatgtca aagggcatgt
tgaggtccaa cagttgaaca accataggtc agtcctcgtc 3180attccggagt
gggaggcaga ggttagccaa aaggtgagag tttgccctgg acgtggctac
3240attctgagag tcactgccta caaggagggc tacggagaag ggtgcgtcac
catccatgaa 3300gttgataaca atactgacca gttgaagttt tccaactgcg
agaaagggca agtgtatcct 3360gggaatacca ttgcatgtaa cgattacaac
aagaatcacg gtgctaacgc ttgctcatct 3420cgcaataggg gatatgatga
gttctatggc aatactccag ctgactactc tgcaaaccag 3480aaagagtatg
gaggagctta caccagccac aaccatgcct atggggaatc ttacgaatcc
3540aacagcagca tcccagcaga ctatgctccg gtctacgagg aggaagccta
cacacatgga 3600aggaggggaa actcatgtga gtacaataga ggctacactc
cgttgccagc tggctacgtc 3660actgccgaat tggagtactt tccggaaacc
gacactgtct gggttgagat aggcgaaact 3720gagggcacct tcatcgttga
caacgtggaa cttctgctta tggaagaata g 37714545PRTBacillus
thuringiensis 4Leu Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala
Val Asn Ala 1 5 10 15 Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys
Thr Asp Val Thr Asp 20 25 30 Tyr His Ile Asp Arg Val Ser Asn Leu
Val Glu Cys Leu Ser Asp Glu 35 40 45 Phe Cys Leu Asp Glu Lys Lys
Glu Leu Ser Glu Lys Val Lys His Ala 50 55 60 Lys Arg Leu Ser Asp
Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg 65 70 75 80 Gly Ile Asn
Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile 85 90 95 Thr
Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu 100 105
110 Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile
115 120 125 Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg
Gly Tyr 130 135 140 Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile
Arg Tyr Asn Ala 145 150 155 160 Lys His Glu Thr Val Asn Val Pro Gly
Thr Gly Ser Leu Trp Pro Leu 165 170 175 Ser Ala Pro Ser Pro Ile Gly
Lys Cys Ala His His Ser His His Phe 180 185 190 Ser Leu Asp Ile Asp
Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly 195 200 205 Val Trp Val
Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu 210 215 220 Gly
Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu 225 230
235 240 Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu
Lys 245 250 255 Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys
Glu Ser Val 260 265 270 Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg
Leu Gln Ala Asp Thr 275 280 285 Asn Ile Ala Met Ile His Ala Ala Asp
Lys Arg Val His Ser Ile Arg 290 295 300 Glu Ala Tyr Leu Pro Glu Leu
Ser Val Ile Pro Gly Val Asn Ala Ala 305 310 315 320 Ile Phe Glu Glu
Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr 325 330 335 Asp Ala
Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser 340 345 350
Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His 355
360 365 Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln
Glu 370 375 380 Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val
Thr Ala Tyr 385 390 395 400 Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr
Ile His Glu Ile Glu Asn 405 410 415 Asn Thr Asp Glu Leu Lys Phe Ser
Asn Cys Val Glu Glu Glu Val Tyr 420 425 430 Pro Asn Asn Thr Val Thr
Cys Asn Asp Tyr Thr Ala Thr Gln Glu Glu 435 440 445 Tyr Glu Gly Thr
Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr 450 455 460 Glu Ser
Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu Glu 465 470 475
480 Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg
485 490 495 Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr
Lys Glu 500 505 510 Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile
Glu Ile Gly Glu 515 520 525 Thr Glu Gly Thr Phe Ile Val Asp Ser Val
Glu Leu Leu Leu Met Glu 530 535 540 Glu 545 51188PRTArtificial
SequenceChimeric protein; DIG3 core toxin with Cry1Ab protoxin 5Met
Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10
15 Ile Pro Thr Val Ser Asn Pro Ser Thr Gln Met Asn Leu Ser Pro Asp
20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Val Ala Glu Val Asn Asn
Ile Asp 35 40 45 Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile
Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe
Ala Gly Gln Leu Ala Ser 65 70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu
Leu Trp Pro Ser Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu
His Val Glu Gln Leu Ile Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr
Arg Asn Thr Ala Ile Ala Arg Leu Glu Gly Leu Gly 115 120 125 Arg Gly
Tyr Arg Ser Tyr Gln Gln Ala Leu Glu Thr Trp Leu Asp Asn 130 135 140
Arg Asn Asp Ala Arg Ser Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala 145
150 155 160 Leu Glu Leu Asp Ile Thr Thr Ala Ile Pro Leu Phe Arg Ile
Arg Asn 165 170 175 Gln Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala
Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe
Gly Ser Glu Trp Gly Thr 195 200 205 Ala Ser Ser Asp Val Asn Gln Tyr
Tyr Gln Glu Gln Ile Arg Tyr Thr 210 215 220 Glu Glu Tyr Ser Asn His
Cys Val Gln Trp Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg
Gly Thr Asn Ala Glu Ser Trp Val Arg Tyr Asn Gln Phe 245 250 255 Arg
Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265
270 Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr
275 280 285 Arg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His Pro Ser
Gln Ala 290 295 300 Phe Ala Ser Thr Thr Trp Phe Asn Asn Asn Ala Pro
Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Val Ile Arg Pro Pro
His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Thr
Leu Ser Arg Trp Ser Asn Thr Gln Phe 340 345 350 Met Asn Ile Trp Ala
Gly His Arg Leu Glu Ser Arg Pro Ile Ala Gly 355 360 365 Ser Leu Asn
Thr Ser Thr Gln Gly Ser Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val
Thr Leu Gln Phe Thr Ser Arg Asp Ile Tyr Arg Thr Glu Ser Leu 385 390
395 400 Ala Gly Leu Asn Ile Phe Ile Thr Gln Pro Val Asn Gly Val Pro
Trp 405 410 415 Val Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg
Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr
Gln Leu Gln Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr
Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser His
Ile Gly Leu Ile Ser Ser Ser His Val 465 470 475 480 Arg Ala Leu Val
Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile
Gly Pro Asn Arg Ile Thr Gln Ile Pro Ala Val Lys Gly Arg 500 505 510
Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515
520 525 Asp Val Val Arg Leu Asn Arg Asn Asn Gly Asn Ile Gln Asn Arg
Gly 530 535 540 Tyr Ile Glu Val Pro Ile Gln Phe Thr Ser Thr Ser Thr
Arg Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Ser
Ile Glu Leu Asn Val Asn 565 570 575 Trp Gly Asn Ser Ser Ile Phe Thr
Asn Thr Leu Pro Ala Thr Ala Ala 580 585 590 Ser Leu Asp Asn Leu Gln
Ser Gly Asp Phe Gly Tyr Val Glu Ile Asn 595 600 605 Asn Ala Phe Thr
Ser Ala Thr Gly Asn Ile Val Gly Val Arg Asn Phe 610 615 620 Ser Ala
Asn Ala Glu Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635
640 Thr Ala Thr Leu Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala
645 650 655 Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys
Thr Asp 660 665 670 Val Thr Asp Tyr His Ile Asp Arg Val Ser Asn Leu
Val Glu Cys Leu 675 680 685 Ser Asp Glu Phe Cys Leu Asp Glu Lys Lys
Glu Leu Ser Glu Lys Val 690 695 700 Lys His Ala Lys Arg Leu Ser Asp
Glu Arg Asn Leu Leu Gln Asp Pro 705 710 715 720 Asn Phe Arg Gly Ile
Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser 725 730 735 Thr Asp Ile
Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr 740 745 750 Val
Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr 755 760
765 Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln Leu
770 775 780 Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu
Ile Arg 785 790 795 800 Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro
Gly Thr Gly Ser Leu 805 810 815 Trp Pro Leu Ser Ala Pro Ser Pro Ile
Gly Lys Cys Ala His His Ser 820 825 830 His His Phe Ser Leu Asp Ile
Asp Val Gly Cys Thr Asp Leu Asn Glu 835 840 845 Asp Leu Gly Val Trp
Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His 850 855 860 Ala Arg Leu
Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly 865 870 875 880
Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys 885
890 895 Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala
Lys 900 905 910 Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp
Arg Leu Gln 915 920 925 Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala
Asp Lys Arg Val His 930 935 940 Ser Ile Arg Glu Ala Tyr Leu Pro Glu
Leu Ser Val Ile Pro Gly Val 945 950 955 960 Asn Ala Ala Ile Phe Glu
Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe 965 970 975 Ser Leu Tyr Asp
Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn 980 985 990 Gly Leu
Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln 995 1000
1005 Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu
1010 1015 1020 Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr
Ile Leu 1025 1030 1035 Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu
Gly Cys Val Thr 1040 1045 1050 Ile His Glu Ile Glu Asn Asn Thr Asp
Glu Leu Lys Phe Ser Asn 1055 1060 1065 Cys Val Glu Glu Glu Val Tyr
Pro Asn Asn Thr Val Thr Cys Asn 1070 1075 1080 Asp Tyr Thr Ala Thr
Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser 1085 1090 1095 Arg Asn Arg
Gly Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val 1100 1105 1110 Pro
Ala Asp Tyr Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp 1115 1120
1125 Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp
1130 1135 1140 Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu
Glu Tyr 1145 1150 1155 Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile
Gly Glu Thr Glu 1160 1165 1170 Gly Thr Phe Ile Val Asp Ser Val Glu
Leu Leu Leu Met Glu Glu 1175 1180 1185 61635DNAArtificial
SequenceSynthetic molecule 6ctcgaggctg aatctgatct cgaaagggca
cagaaagctg taaacgcatt gtttacaagt 60tctaatcaaa tcggactcaa aaccgatgtt
acggactatc acatagatag ggtttctaat 120cttgtggaat gtctttcaga
tgagttttgt ttagatgaga agaaagaact ttcagaaaag 180gtcaagcacg
ccaaaagact gtccgatgaa aggaatctcc ttcaagaccc aaactttcgt
240ggaatcaata ggcagctcga cagaggttgg agagggagca cagatatcac
cattcaagga 300ggagatgacg ttttcaaaga gaactatgtc accttgttag
gcacctttga tgagtgctat 360ccaacttatc tgtatcagaa gattgatgaa
tccaagctga aggcttacac aagatatcag 420ctcagaggat acatcgagga
ctcccaagat ttggagatat acttgattcg ttacaatgca 480aaacatgaga
ccgtgaatgt tcctggtact ggaagtctct ggccactgtc tgctccgtca
540cctattggga aatgtgccca tcactcccac catttctcat tggacataga
cgttggctgc 600acagatttga atgaagattt gggtgtttgg gtcatcttca
agatcaaaac tcaagacgga 660cacgctcgtt taggaaactt agagtttctt
gaagagaagc ccttggttgg ggaggcactt 720gccagagtaa agagagctga
aaagaagtgg agagataaga gggagaaact tgagtgggag 780actaacattg
tgtacaagga agccaaagaa agcgtggatg ctcttttcgt gaactctcag
840tatgataggt tacaagcaga caccaacata gcaatgatac atgcagctga
caaaagagtc 900cattctattc gtgaggctta cttgccagaa cttagtgtga
ttcccggtgt caacgctgcc 960attttcgagg aattggaagg aagaatcttt
acggctttca gcctctatga cgctaggaat 1020gttatcaaga atggtgattt
caacaatggc ctctcatgtt ggaatgtgaa aggtcatgtt 1080gatgtagagg
agcaaaacaa tcaccgtagc gtgctggttg tcccagaatg ggaagccgaa
1140gtaagccaag aagttagagt ttgccctgga agaggctaca ttctgcgtgt
caccgcttac 1200aaagaaggat atggcgaagg gtgcgtgact attcatgaga
ttgagaacaa tactgacgaa 1260cttaagtttt caaactgcgt cgaggaggaa
gtgtatccta acaacacagt gacttgtaat 1320gactatacag caacgcaaga
ggaatacgag gggacataca ccagtcgtaa tcgtggttat 1380gatggtgctt
atgaaagcaa ttcatccgtt ccagctgact atgccagtgc ctacgaagag
1440aaggcttaca cggatggcag aagagataac ccatgtgagt ccaacagagg
ttatggtgat 1500tacactcctc ttccagctgg ttacgtgact aaagagttag
agtactttcc ggagactgat 1560aaggtttgga ttgaaatcgg agagacagaa
gggacattca tagtagattc agttgagctt 1620cttctcatgg aagaa
163571635DNAArtificial SequenceSynthetic molecule 7ctcgaggctg
aatcggatct tgaaagggca cagaaggcag tcaacgctct cttcaccagc 60tcaaatcaga
ttggccttaa gaccgatgtt actgactatc atatcgacag agtttctaac
120cttgtcgagt gcctctccga cgagttctgt ctcgacgaaa agaaggaact
ctccgagaaa 180gtgaagcacg cgaaacgcct ctcggatgaa cggaacttgc
tgcaagatcc gaacttcaga 240ggcatcaatc gccagttgga tagaggctgg
aggggatcaa ccgacataac cattcaaggt 300ggggatgatg tgttcaagga
aaactacgtg acattgctgg gcaccttcga cgagtgctat 360cccacgtatc
tctatcagaa gattgacgag tccaagctca aagcctacac acgctatcag
420ctcagaggct acattgagga ctctcaagac ctcgaaatct acttgatcag
atacaacgcc 480aagcacgaga cggtgaacgt ccctgggact gggtcactgt
ggccactgtc ggcaccctcg 540ccaatcggaa agtgcgctca ccacagccac
cacttctccc ttgacataga tgttgggtgt 600acggacttga atgaggatct
gggtgtgtgg gtgatcttta agatcaagac ccaagatggt 660catgcgaggc
ttggcaacct tgagttcctt gaagagaagc ctttggtcgg agaggcactg
720gctcgcgtga agagggctga gaagaaatgg agggacaaga gggagaaact
ggagtgggag 780accaacatag tgtacaagga ggccaaggag tcagtggacg
cactgtttgt caattcccag 840tatgataggc tccaagcgga cacgaacatc
gccatgatcc
atgcagcgga caagagggtt 900cactccataa gggaggccta tcttccggag
ctgtcagtga ttcctggggt caacgcagcc 960atctttgagg aattggaagg
gaggatcttc accgctttct ctctgtacga cgctcggaac 1020gtcatcaaga
atggtgattt caacaatgga ctcagctgct ggaacgtgaa agggcatgtc
1080gatgttgaag aacagaacaa tcaccgcagc gtgctggtgg ttccggagtg
ggaagccgag 1140gtctcacaag aagtcagagt gtgccctggg aggggttaca
tcttgcgggt cacagcctac 1200aaggaaggtt atggcgaagg ctgtgtcacg
atccatgaga tcgaaaacaa cacagacgag 1260ctgaagtttt ccaactgtgt
tgaggaggag gtctatccta acaatactgt tacgtgcaac 1320gactacacag
ccactcaaga ggagtacgag ggcacttaca cctctcgcaa cagaggctac
1380gacggtgcct acgagtcaaa cagctccgtg ccagcggact acgcctcggc
ttacgaagag 1440aaggcgtaca ccgacggtcg gagggataac ccgtgcgaga
gcaatagagg ctatggcgac 1500tacactcctc tcccagctgg ctacgtgacc
aaggagttgg agtactttcc ggagacagac 1560aaagtctgga ttgagattgg
agagacagaa ggcacgttca tcgtggactc tgttgaactc 1620ttgctgatgg aggag
1635
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References